SR-BI in bone marrow derived cells protects mice from diet induced coronary artery atherosclerosis and myocardial infarction.

SR-BI deficient mice that are also hypomorphic for apolipoprotein E expression develop diet induced occlusive coronary artery atherosclerosis, myocardial infarction and early death. To test the role of SR-BI in bone marrow derived cells, we used bone marrow transplantation to generate SR-BI-null; apoE-hypomorphic mice in which SR-BI expression was restored solely in bone marrow derived cells. SR-BI-null; apoE-hypomorphic mice were transplanted with SR-BI(+/+)apoE-hypomorphic, or control, autologous SR-BI-null; apoE-hypomorphic bone marrow. Four weeks later, mice were fed a high-fat, high-cholesterol, cholate-containing diet to induce coronary artery atherosclerosis. Mice transplanted with autologous bone marrow developed extensive aortic atherosclerosis and severe occlusive coronary artery atherosclerosis after 4 weeks of feeding. This was accompanied by myocardial fibrosis and increased heart weights. In contrast, restoration of SR-BI expression in bone marrow derived-cells reduced diet induced aortic and coronary artery atherosclerosis, myocardial fibrosis and the increase in heart weights in SR-BI-null; apoE-hypomorphic mice. Restoration of SR-BI in bone marrow derived cells did not, however, affect steady state lipoprotein cholesterol levels, but did reduce plasma levels of IL-6. Monocytes from SR-BI-null mice exhibited a greater capacity to bind to VCAM-1 and ICAM-1 than those from SR-BI(+/+) mice. Furthermore, restoration of SR-BI expression in bone marrow derived cells attenuated monocyte recruitment into atherosclerotic plaques in mice fed high fat, high cholesterol cholate containing diet. These data demonstrate directly that SR-BI in bone marrow-derived cells protects against both aortic and CA atherosclerosis

Restoration of SR-BI expression in BM derived cells did not alter lipids in HFCC diet fed SR-BI-null/apoE-hypomorphic mice.

<p><b>A.</b> Total cholesterol, <b>B.</b> unesterified (free) cholesterol and <b>C.</b> triglycerides were measured in plasma collected from fasted SR-BI^−/− → SR-BI^−/− (white bars) and SR-BI^+/+ → SR-BI^−/− mice (black bars) fed the HFCC diet for 3 weeks (n = 6 mice/group). <b>D.</b> Averaged lipoprotein total cholesterol profiles from n = 10 SR-BI^−/− → SR-BI^−/− (white symbols) and SR-BI^+/+ → SR-BI^−/− mice (black symbols) fed the HFCC diet for 3 weeks. Fractions corresponding to the elution of purified human VLDL, IDL/LDL and HDL are shown at the top. <b>E.</b> Total cholesterol fractions containing VLDL-sized (1–14), IDL/LDL-sized (15–28) and HDL-sized lipoproteins (29–42) from the lipoprotein total cholesterol profiles of individual mice were determined. Averages ± standard errors are shown (n = 10 mice/group). No statistically significant differences in values from SR-BI^−/− → SR-BI^−/− (white bars) and SR-BI^+/+ → SR-BI^−/− mice (black bars) were detected.</p

Knockout of SR-BI in macrophages does not affect AcLDL driven foam cell formation.

<p>Resident peritoneal macrophages collected from SR-BI^+/+apoE-hypomorphic or SR-BI^−/−apoE-hypomorphic mice were cultured in the absence or presence of 100 µg/ml AcLDL for 24 hrs, fixed and stained with oil red O (red) and DAPI (blue). <b>A</b>–<b>D</b>: Representative fluorescence images. Scale bar  =  100 µm. <b>E</b>. Quantification of lipid accumulation as the oil red O staining intensity normalized to the number of DAPI stained nuclei per field of view. Five fields of view were analyzed per sample. Data are averages ± standard errors of triplicate samples. The experiment was performed three times and a representative result is shown. Oil red O staining intensities for AcLDL treated versus untreated cells were statistically significantly different but staining intensities for AcLDL treated SR-BI^+/+ and SR-BI^−/− cells were not (determined by Student’s T test).</p

Restoration of SR-BI expression in BM derived cells attenuates diet induced increases in heart size and cardiac fibrosis in SR-BI-null/apoE-hypomorphic mice.

<p><b>A.</b> Ratios of heart weight to body weight (HW:BW) are plotted for SR-BI^−/− → SR-BI^−/− mice (circles) and SR-BI^+/+ → SR-BI^−/− mice (diamonds) fed the HFCC diet for 4 weeks. Red symbols identify those SR-BI^−/− → SR-BI^−/− mice which appeared to be in poor health or were moribund at collection. Closed symbols denote males and open symbols denote females. Horizontal bars indicate the mean heart/body weight ratios (black bars  =  males, open bars  =  females, blue bars  =  pooled males and females). Data passed the Shapiro-Wilk test for normality and was analyzed by the Student’s T test; P<0.0006 for SR-BI^−/− → SR-BI^−/− vs SR-BI^+/+ → SR-BI^−/− mice. <b>B.</b> Body weights from the same mice as in A are plotted for male (closed symbols) and female (open symbols) SR-BI^−/− → SR-BI^−/− (circles) and SR-BI^+/+ → SR-BI^−/− (diamonds). Bars denote average body weights: black bars  =  males, open bars  =  females; blue bars  =  pooled male + female mice. Differences in body weights between males (P = 0.006) and when males and females were pooled (P = 0.03) were significant, but females were not, by Student’s T test. <b>C.</b> Appearance of hearts from SR-BI^−/− → SR-BI^−/− and SR-BI^+/+ → SR-BI^−/− mice after 4 weeks of HFCC diet feeding. <b>D–E.</b> Trichrome staining of cross sections of hearts from SR-BI^−/− → SR-BI^−/− (D) and SR-BI^+/+ → SR-BI^−/− mice (E) after 4 weeks of HFCC diet feeding. Healthy myocardium appears red while collagen-rich fibrotic areas appear blue. L indicates the lumen of the left ventricle. Representative images are shown in C–E. Scale bars  =  300 µm.</p

Effects of restoring SR-BI expression in BM derived cells on plasma levels of TNFα and IL-6 in HFCC diet fed SR-BI-null/apoE-hypomorphic mice.

<p><b>A.</b> TNFα and <b>B.</b> IL-6 levels in plasma from control SR-BI^−/− → SR-BI^−/− (white bars) and SR-BI^+/+ → SR-BI^−/− mice (black bars) after 3 weeks of HFCC diet feeding. Shown are mean levels for n = 8 mice per group. Error bars correspond to standard errors. <b>A.</b> TNFα levels were not statistically significantly different. <b>B.</b> IL-6 levels were statistically significantly different. P = 0.028. Statistical analysis was by the Mann-Whitney rank sum test.</p

SR-BI attenuates monocyte recruitment.

<p><b>A.</b> ICAM-1 binding, <b>B.</b> VCAM-1 binding, by monocytes from SR-BI^+/+apoE-hypomorphic (blue) or SR-BI^−/−apoE-hypomorphic mice (red) as determined by flow cytometry. Representative histograms of 4 replicates are shown. <b>C</b>–<b>G</b>: <i>In vivo</i> recruitment of latex bead marked monocytes into atherosclerotic plaques. Fluorescence images in dark-field (C and E) or overlayed onto brightfield images (D and F) of oil red O stained aortic sinus atherosclerotic plaques from SR-BI^−/− → SR-BI^−/− (C, D) and SR-BI^+/+ → SR-BI^−/− mice (E, F). Arrows in D and F indicate the positions of green fluorescent latex bead marked cells. Scale bar  =  50 µm. G. Quantification of fluorescent bead marked cells in atherosclerotic plaques (normalized to plaque sizes). Data are averages ± standard errors. N = 4 mice per group. P = 0.039 by Student’s T test.</p

Restoration of SR-BI expression in BM derived cells by BM transplantation reduces diet induced atherosclerosis in SR-BI-null/apoE-hypomorphic mice.

<p>SR-BI^−/−apoE-hypomorphic mice, at 10 weeks of age, were transplanted with BM from either control SR-BI^−/− or SR-BI^+/+ mice to generate SR-BI^−/− → SR-BI^−/− (circles) and SR-BI^+/+→ SR-BI^−/− mice (diamonds). BM transplanted mice were fed a high fat, high cholesterol, cholate containing (HFCC) diet for four weeks. <b>A–C:</b> Aortic sinus atherosclerosis. Representative oil red O and hematoxylin stained sections of the aortic sinus are shown in panels <b>A</b> and <b>B</b> for mice transplanted with BM from SR-BI^−/− or SR-BI^+/+ donors. Scale bars = 100 µm. <b>C.</b> Atherosclerotic plaque sizes were quantified for n = 17 SR-BI^−/− → SR-BI^−/− and n = 13 SR-BI^+/+→ SR-BI^−/− mice. Donor BM is indicated. P<0.001 by the Mann Witney rank sum test. <b>D–F:</b> Coronary artery atherosclerosis. Representative images of oil red O and hematoxylin stained coronary artery sections are shown in panels <b>D</b> and <b>E.</b> Scale bars = 50 µm. <b>F.</b> Coronary arteries in heart sections were scored as “occluded” if they contained raised atherosclerotic plaques and the proportions of occluded coronary arteries are plotted. Group sizes are as for Panel C. P = 3×10⁻⁹ by Student’s T-test and P<0.001 by the Mann Witney rank sum test. For panels C and F, male and female mice are indicated by closed and open symbols respectively. Averages are indicated by bars: black bars  =  males, open bars  =  females, blue bars  =  pooled males and females. Data for males vs females were not statistically significantly different. Immunostaining (red) for SR-BI (G, H) or CD68 (<b>I, J</b>) in atherosclerotic plaques from a separate group of female SR-BI^−/− → SR-BI^−/− mice (<b>G, I</b>) and SR-BI^+/+→ SR-BI^−/− mice (<b>H, J</b>) fed the HFCC diet for 18 days. Representative images are shown. Yellow arrow in G points to SR-BI negative cells; yellow arrows in H-J indicate cells positive for SR-BI or CD68. Nuclear DNA was stained with DAPI. Scale bars = 100 µm. <b>K.</b> Quantification of CD68 staining area in n = 6 mice per group (all females). Data are means ± standard errors. P = 0.039 by Student’s T-test.</p