Hepatic gene expression levels and circulating IL-6 levels.

<p>mRNA expression of TNFα (A), and IL-6 (B) in liver from bone marrow transplanted LDLr KO mice. Gene expression analysis was performed by RT-PCR. Values are relative to the average expression of housekeeping genes and expressed as fold induction compared to WT transplanted controls. Values are mean ± SEM, n≥3 mice per group as compared to WT. Increased circulating IL-6 levels in LDLr KO mice transplanted with ABCA1/apoE double KO bone marrow (C). Serum from the transplanted LDLr KO mice was collected at 17 weeks after transplantation including 9 weeks of WTD feeding. Values are mean ± SEM, n≥8 mice per group. *p<0.05, **p<0.01. ***p<0.001 as compared to WT transplanted controls. ^#p<0.05 as compared to ABCA1 KO transplanted animals.</p

Successful disruption of macrophage ABCA1 and apoE by bone marrow transplantation.

<p>RT-PCR on genomic DNA of bone marrow isolated from ABCA1 and/or apoE KO transplanted LDLr KO mice and their WT transplanted controls at 17 weeks after transplantation.</p

<i>In vitro</i> cellular cholesterol efflux.

<p>Cholesterol efflux from bone marrow-derived macrophages (BMDM) to BSA (A), apoA-I (10 µg/mL; B) and HDL (50 µg/mL; C) was determined after 24 h. Data are expressed as the percentage of radioactivity released into the medium. Values represent the means of n≥3 mice ± SEM per group. ***p<0.001 as compared to WT transplanted controls.</p

Analysis of atherosclerosis in the aortic arch of bone marrow transplanted LDLr KO mice after 9 weeks of HF/HC diet feeding.

<p>Relative plaque area in the aortic arch was quantified <i>en face</i> calculating the ratio of atherosclerotic lesions to the surface of the entire aortic arch (left panel). Representative pictures (right panels). **P<0.01 as compared to WT transplanted controls.</p

Lipoprotein profiles from ABCA1 and/or apoE KO transplanted LDLr KO mice and their WT transplanted controls.

<p>Fifty µL samples of serum, pooled from 2 mice per genotype, collected after 9 weeks of HF/HC feeding, were subjected to FPLC fractionation. Distribution of serum FC and TC over the fractions (25 µL) was determined enzymatically. Values represent the means of n≥5 samples per group. 1 sample corresponds to pooled serum of 2 mice.</p

Analysis of atherosclerosis in the aortic root of bone marrow transplanted LDLr KO mice after 9 weeks of HF/HC diet feeding.

<p>Quantification of atherosclerotic lesion sizes in the aortic root and TC levels in bone marrow transplanted LDLr KO mice after 9 weeks of HF/HC diet feeding (A). Lesion size divided by TC levels show cholesterol independent effects on lesion development (B). Representative cross-sections, stained with oil red-O (magnification 50×; lower panels).Values are the means of 10 aortic root sections of individual mice (n≥11 mice ± SEM per group. **p<0.01, ***p<0.001 as compared to WT transplanted controls, ^#p<0.05 as compared to ABCA1 KO transplanted animals.</p

ApoE protein content in VLDL and LDL fractions from ABCA1 and/or apoE KO transplanted LDLr KO mice and their WT transplanted controls.

<p>Western blots showing apoE protein expression in VLDL (A) and LDL (B) isolated from ABCA1 KO and/or apoE KO transplanted LDLr KO animals and WT transplanted controls after feeding the HF/HC diet for 9 weeks. Samples were adjusted for cholesterol content. The density of the bands was determined using quantification software (Image J). n≥5 samples per group (pooled). WT values were set at 100%.</p

Effects of macrophage ABCA7 deficiency on plasma lipid levels, atherosclerosis and ABCA1 expression in peritoneal macrophages from LDLr KO mice reconstituted with WT and ABCA7 KO bone marrow.

<p>(<b>A</b>) Blood samples were drawn after an overnight fast. The concentrations of cholesterol, phospholipids and triglycerides in serum were determined using enzymatic colorimetric assays. (<b>B</b>) The mean lesion area (µm²) was calculated from Oil red O-stained cross-sections of the aortic root at the level of the tricuspid valves. (<b>C</b>) Guanidium thiocyanate-phenol was used to extract total RNA from cells and mRNA expression was determined by real-time RT-PCR. Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT), β-actin, and acidic ribosomal phosphoprotein PO (36B4) were used as the standard housekeeping genes. Relative gene expression was calculated by subtracting the threshold cycle number (Ct) of the target gene from the average Ct of housekeeping genes and raising two to the power of this difference. Values represent the mean ± SEM of ≥6 mice per group. Statistically significant difference *p<0.05.</p

Serum cholesterol levels in WT, ABCA1 KO, ABCA7 KO and dKO bone marrow transplanted mice on chow and WTD.

<p>Serum cholesterol levels were measured in LDLr^−/− at 8 and 18 weeks after transplantation with bone marrow from WT, ABCA1 KO, ABCA7, or dKO mice. At 8 weeks after bone marrow transplantation, the regular chow diet was switched to a high-cholesterol diet. Data represent the means±SEM of 12–16 mice. Statistical significance of *p<0.05, **p<0.01, and ***p<0.001 compared with WT transplanted mice. Abbreviations: WTD = Western-type diet; VLDL = very-low-density lipoprotein; LDL = low-density lipoprotein; HDL = high-density lipoprotein; C = cholesterol.</p

Effect of macrophage ABCA1, ABCA7, and combined ABCA1/ABCA7 deficiency on <i>in vivo</i> foam cell formation, and mRNA and protein expression levels of ABCA1 and ABCG1.

<p>(<b>A</b>) Peritoneal leukocytes were analyzed using a hematology Sysmex XT-2000iV analyzer and the number of macrophage foam cells were quantified as percentage of the total amount of isolated cells. Values represent the mean±SEM of ≥5 mice per group. Statistically significant difference **p<0.01, and ***p<0.001 vs. WT. (<b>B</b>) Photomicrographs of oil-red O-stained cytospins of peritoneal cells. Original magnification ×400. (<b>C</b>) Lipids were extracted from peritoneal leukocytes with organic solvents. (<b>D</b>) Guanidium thiocyanate-phenol was used to extract total RNA from peritoneal macrophages and mRNA expression was determined by real-time RT-PCR. Hypoxanthine Guanine Phosphoribosyl Transferase (HPRT), β-actin, and acidic ribosomal phosphoprotein PO (36B4) were used as the standard housekeeping genes. Relative gene expression was calculated by subtracting the threshold cycle number (Ct) of the target gene from the average Ct of housekeeping genes and raising two to the power of this difference. WT samples were all calibrated to an arbitrary unit. (<b>E</b>) Western-blot analyses were performed to quantify the protein expression levels of ABCA1 and ABCG1 in peritoneal leukocytes. Values represent the mean ± SEM of 4–6 mice per group. Statistically significant difference *p<0.05, **p<0.01 and ***p<0.001.</p