Figure 1.
PLTP production by mouse macrophages.
Peritoneal macrophages were collected from either C57BL/6J wild type (wt/wt), hemizygous huPLTP transgenic (tg/wt) and homozygous huPLTP transgenic (tg/tg) mice and kept in culture for 24 hours. Subsequently, PLTP activity was measured in a sample of the culture medium. PLTP activity is expressed as arbitrary units (AU; 1 AU is equivalent to 14 µmol/mL/h). N = cells from 6 mice per group. ** p<0.005 versus wt/wt; $ p<0.01 versus tg/wt.
Figure 2.
A. PLTP activity was measured in plasma samples and expressed in arbitrary units as described in the legend of Fig. 1. Left panel: plasma samples were collected from non-transplanted LDLR−/− and LDLR−/−/huPLTPtg/wt mice just before the start of the high fat, high cholesterol diet (0 weeks, white bars) and after 9 weeks of the diet (9 weeks, black bars). *** p<0.001 versus 0 weeks (same genotype), ### p<0.001 versus LDLR−/− mice (on the same diet). Right panel: plasma samples were collected from PLTPwt/wt→LDLR−/−, huPLTPtg/wt→LDLR−/−, and huPLTPtg/tg→LDLR−/− recipient mice at 1 week before the start of the transplantation procedure (i.e. at 10 weeks before the start of the diet: −10 weeks, grey bars), just before the start of the diet (0 weeks, white bars) and after 9 weeks of diet (9 weeks, black bars). *** p<0.001 versus −10 weeks (same genotype); ### p<0.001 versus PLTPwt/wt→LDLR−/− mice (on the same diet); ¶¶¶ p<0.001 versus 0 weeks (same genotype). B. Mass of human PLTP in plasma from mice was measured by ELISA as described in Methods in the Supplemental Data. C. Specific Activities of PLTP were calculated using the ratio between the activity in AU and the mass in mg/L. ¶¶¶ p<0.001 versus 0 weeks (same genotype); $$$ p<0.001 versus PLTPtg/wt→LDLR−/− mice (on the same diet). N = 11–15 mice per group.
Figure 3.
Plasma levels of cholesterol (A), phospholipids (B) and triglycerides (C) were measured as described in Methods. Left panels: Plasma samples were collected from non-transplanted LDLR−/− and LDLR−/−/huPLTPtg/wt mice just before the start of the high fat, high cholesterol diet (0 weeks, white bars) and after 9 weeks of the diet (9 weeks, black bars). *** p<0.001 versus 0 weeks (same genotype), ### p<0.001 versus LDLR−/− mice (on the same diet). Right panels: plasma samples were collected from PLTPwt/wt→LDLR−/−, huPLTPtg/wt→LDLR−/−, and huPLTPtg/tg→LDLR−/− recipient mice at 1 week before the start of the transplantation procedure (i.e. at 10 weeks before the start of the diet: −10 weeks, grey bars), just before the start of the diet (0 weeks, white bars) and after 9 weeks of diet (9 weeks, black bars). N = 11–15 mice per group. * p<0.05, *** p<0.001 versus −10 weeks (same genotype); ### p<0.001 versus PLTPwt/wt→LDLR−/− mice (on the same diet).
Figure 4.
Plasma levels of non-HDL cholesterol and HDL cholesterol.
Plasma was collected from PLTPwt/wt→LDLR−/−, huPLTPtg/wt→LDLR−/−, and huPLTPtg/tg→LDLR−/− recipient mice at the end of the high fat, high cholesterol diet period (i.e., at time of sacrifice) and separated into two fractions: d<1.063 g/L (non-HDL) and d>1.063 g/L (HDL) by ultracentrifugation. In both fractions, the cholesterol concentration was measured. A: non-HDL cholesterol; B: HDL-cholesterol. N = 11–15 mice per group. ** p<0.01, *** p<0.001 versus PLTPwt/wt→LDLR−/− mice; $$$ p<0.001 versus huPLTPtg/wt→LDLR−/− mice.
Figure 5.
Atherosclerosis in non-transplanted and transplanted mice.
A. Plaque area was measured in sections from the aortic root (see Methods). Left panel: Plaque area in non-transplanted LDLR−/− and LDLR−/−/huPLTPtg/wt mice. *** p<0.001 versus LDLR−/− mice. Right panel: Plaque area in PLTPwt/wt→LDLR−/−, huPLTPtg/wt→LDLR−/−, and huPLTPtg/tg→LDLR−/− recipient mice. N = 11–15 mice per group. *** p<0.001 versus PLTPwt/wt→LDLR−/− mice; $$$ *** p<0.001 versus huPLTPtg/wt→LDLR−/− mice. B, C. Immunohistochemistry of atherosclerotic lesions from non-transplanted LDLR−/−/huPLTPtg/wt mice (B) and from transplanted huPLTPtg/tg→LDLR−/− mice (C), stained for macrophages (MΦ) with anti-CD68 and for PLTP and counterstained with Nuclear-fast red (serial sections). The right panels are magnifications of the boxed parts in the left panels. Original magnifications: 100x (B) and 250x (C).
Figure 6.
Transplantations with β-actin GFP mice→LDLR−/− mice.
A–D: Early lesions (A,B) and advanced lesions (C,D) with donor cells expressing GFP (green), CD 68 (marker for macrophages) in red (A,C) and α-actin (marker for VSMCs) in red (B,D) and nuclei stained with DAPI (blue). Endothelial cells covering the lesions do not express GFP (nuclei indicated with arrows). Co-localization of GFP and CD68 results in an orange color (arrowheads). The necrotic core in the advanced lesion (located centrally in C and D) is diffusely positive for CD68 but does not show any GFP signal. The media is marked with a double arrow (↔). E,F: Early lesion (E) and advanced lesion (F) with donor cells expressing GFP (green), and CD 31 (marker for endothelial cells) in red (arrows). Representative pictures from N = 6 animals are shown. Original magnifications: 200X.
Figure 7.
Characteristics of mouse macrophages.
A. Cholesterol efflux from cultured peritoneal macrophages. Cells were loaded with radioactive cholesterol as described in Methods. Subsequently, culture medium with human HDL as an acceptor was added. Left panel: Radioactivity was measured in aliquots from the cultured medium taken at 0, 1, 2, 4, and 6 hours. Circles: macrophages from C57BL/6J mice, squares: macrophages from huPLTPtg/tg mice. Differences were not statistically significant. B. PLTP activity measured in the medium of macrophages cultured for 24 hours in the absence (white bars) or presence (black bars) of 100 ng/mL LPS. Cells from 6 mice were used per group. C. PLTP activity measured in plasma samples from mice treated with thioglycolate. N = 6 mice per group. ** p<0.005, *** p<0.001 versus cells without LPS or thioglycolate (same genotype), ## p<0.005, ### p<0.001 versus PLPTwt/wt (same culture medium, with or without LPS or thioglycolate).