HDL stimulated macrophage migration involves MAP kinase and PKC pathways.

<p>RAW 264.7 cells were cultured in media containing 3%NCLPDS for 18 hrs. Cells were pre-incubated with the indicated inhibitors for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. Control cells were treated with DMSO vehicle. <b>A.</b> Cells were treated with either 10 µM of the ERK1/2 pathway inhibitor, PD98059, or 1 µM of the p38 MAPK inhibitor, SB230580. <b>B.</b> Cells were treated with 5 µM of the PKC inhibitors Ro31-8220 or Go6976. Data are means ± standard deviations of 3 replicates. Values identified with different letters are statistically significantly different (P<0.04, 2 way ANOVA with Tukey post hoc test).</p

HDL stimulates the migration of macrophages but not of lipid loaded macrophage foam cells.

<p><b>A.</b> Wild type mouse peritoneal macrophages were incubated in lipoprotein deficient serum overnight and cell migration in response to no stimulus (control), apoA1(100 µg/ml), HDL (100 µg protein/ml) or MCP-1 (100 ng/ml) (each added to the bottom well of the migration assay chamber) was performed as described in the Methods section. The number of migrated cells/well from three independent samples from each group is represented as the mean ± standard deviation. <b>B.</b> Wild type mouse peritoneal macrophages, or <b>C.</b> RAW 264.7 cells were incubated in collagen I coated cell culture dishes with either HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) under conditions paralleling the migration assay. Cell adhesion was measured by counting DAPI stained nuclei. The degree of cell adhesion was normalized to that in control cells and is represented as the mean ± standard deviation of triplicates. <b>D.</b> Foam cells from wild type mouse peritoneal macrophages were generated by culture for 48 hrs in the presence of AcLDL (100 µg/ml). Cells were washed and migration in response to HDL was measured as described in the Methods section. The number of migrated cells/well is plotted as the mean ± standard deviation of triplicates. Statistical analysis was done using one way ANOVA with Holm-Sidak post hoc test (A–C) or Student's T-test (D). Values identified with different letters are statistically significantly different (P<0.05). NS indicates that no statistically significant difference was detected.</p

Working model for HDL mediated stimulation of macrophage migration.

<p>HDL binding to SR-BI leads to activation of S1PR1 signaling. This may involve transfer of S1P from bound HDL to S1PR1. Inhibition of HDL binding to SR-BI (blocking antibody) or SR-BI-mediated lipid transfer (BLT-1) prevents HDL dependent activation of S1PR1 signaling, but does not affect direct activation of S1PR1 by agonists. Inactivation of expression of SR-BI or PDZK1, on the other hand, inhibits migratory responses to FTY720, through an as yet unknown mechanism. HDL dependent activation of migration is suppressed by inhibition of S1PR1 signaling with FTY720 or W146, which directly antagonize S1PR1, or with PTX, which blocks Gα_i coupled GPCR's including CCR2 (receptor for MCP-1). Upon appropriate stimulation, S1PR1 (and CCR2) stimulate macrophage migration by activation of diverse signaling pathways including PI3K/Akt1, Rho kinase, PKC, p38 MAPK and Erk1/2 pathways .</p

HDL-induced migration is reduced in macrophages deficient in either SR-BI or PDZK1.

<p><b>A.</b> Peritoneal macrophages were prepared from either wild type (WT) or SR-BI KO mice, and cultured in the presence of 3% NCLPDS for 16 hrs before analysis. Migration of cells in response to no stimulus (control), HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106487#pone-0106487-g001" target="_blank">Figure 1</a> and the Methods section. <b>B.</b> Peritoneal macrophages from WT or SR-BI KO mice were cultured as described in A, prior to incubation with or without HDL (100 µg protein/ml) for the times indicated. Actin filaments were visualized by fluorescence microscopy after alexa 488-phalloidin staining. Representative images are shown. Scale bars = 10 µm. <b>C.</b> Numbers of cells with lamellipodia (arrows in B) were counted (∼100 cells over 4–5 fields) for cells isolated from three mice from each genotype. <b>D.</b> Migration of peritoneal macrophages from WT or PDZK1 KO mice as described for panel A. <b>E.</b> Flow cytometry analysis of cell surface SR-BI levels in wild type, SR-BI KO and PDZK1 KO macrophages. Values in A, C and D are means ± standard deviations of triplicates and are representative of multiple independent assays. Data in E is representative of multiple analyses. Statistical analysis in panels A, C and D was done using two way ANOVA with Tukey post hoc test. Values identified with different letters are statistically significantly different (P<0.001). NS indicates that no statistically significant difference was detected.</p

HDL stimulated macrophage migration involves Rho kinase and PI3K-Akt 1 signaling.

<p><b>A.</b> RAW 264.7 cells were cultured in media containing 3% NCLPDS for 18 hrs. Cells were pre-incubated with 10 µM of either the Rho Kinase inhibitor, Y-27632, or the PI3K inhibitor LY294002, or with DMSO vehicle for 30 min and then the migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence or absence of the indicated inhibitors. <b>B.</b> RAW 264.7 cells were serum starved for 18 hrs, washed and treated with or without HDL (100 µg protein/ml) for 10, 30 or 60 min. Equal amounts of proteins were analyzed by SDS-PAGE and immunoblotting for either phospho-Ser473- or total-Akt. <b>C.</b> MPM's were harvested from 6 independent WT, Akt1 KO, Akt2 KO or Akt3 KO mice. Migration in response to no stimulus, HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured. Data from A and C are means ± standard deviations of 6 replicates done over two independent experiments. Values identified with different letters are statistically significantly different (P<0.003, 2 way ANOVA with Tukey post hoc test). <b>D.</b> Flow cytometry analysis of cell surface SR-BI levels in wild type, SR-BI KO and Akt1 KO macrophages. Shown are representative histograms of an experiment performed twice.</p

Antibody or small molecule mediated inactivation of SR-BI inhibits HDL dependent but not FTY720 dependent macrophage migration.

<p>Mouse peritoneal macrophages were pretreated for 30 min with either <b>A.</b> An anti-SR-BI blocking antiserum or pre-immune rabbit serum (0.5 µg/ml); or <b>B.</b> BLT-1 (0.3 µM), an inhibitor of SR-BI mediated lipid transfer, or DMSO vehicle control. Cell migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml) or MCP-1 (100 ng/ml) was measured in the continued presence of the inhibitors as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106487#pone-0106487-g001" target="_blank">Figure 1</a> and the Methods section. Data are means ± standard deviations of 6 replicates. Statistical analysis was done using two way ANOVA with Tukey post hoc test. Values identified with different letters are statistically significantly different (P<0.002).</p

Blockade of S1PR1 prevents HDL stimulated macrophage migration.

<p>RAW 264.7 (panels A,B) or mouse peritoneal macrophages (panel D) were pretreated for 30 min with the following: <b>A.</b> PTX (100 ng/ml) an inhibitor of Gα_i protein coupled receptors; <b>B.</b> VPC23019 (10 µM) an antagonist of S1PR's 1, and 3; or <b>D.</b> W-146 (10 µM) an antagonist specific for S1PR1. Control cells were treated with either 2 µg/ml BSA or DMSO vehicle as indicated. Cell migration in response to HDL (100 µg protein/ml), FTY720 (2 ng/ml), MCP-1 (100 ng/ml) and/or SEW2871 (5 nM) was measured in the continued absence or presence of the inhibitors as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106487#pone-0106487-g001" target="_blank">Figure 1</a> and the Methods section. <b>C.</b> Analysis of expression of S1PR1, 2 and 3 in peritoneal macrophages (left panel) or lung tissue (right panel) from wild type mice. Data are means ± standard deviations of 6 replicates (A,B) or 3 replicates (C,D). Statistical analysis was done using two way ANOVA with Tukey post hoc test. Values identified with different letters are statistically significantly different (P<0.002).</p

Modifications in Perfringolysin O Domain 4 Alter the Cholesterol Concentration Threshold Required for Binding

Changes in the cholesterol content of cell membranes affect many physiological and pathological events, including the formation of arterial plaques, the entry of virus into cells, and receptor organization. Measuring the trafficking and distribution of cholesterol is essential to understanding how cells regulate sterol levels in membranes. Perfringolysin O (PFO) is a cytolysin secreted by <i>Clostridium perfringens</i> that requires cholesterol in the target membrane for binding. The specificity of PFO for high levels of cholesterol makes the toxin an attractive tool for studying the distribution and trafficking of cholesterol in cells. However, the use of the native toxin is limited given that binding is triggered only above a determined cholesterol concentration. To this end, we have identified mutations in PFO that altered the threshold for how much cholesterol is required to trigger binding. The cholesterol threshold among different PFO derivatives varied up to 10 mol % sterol, and these variations were not dependent on the lipid composition of the membrane. We characterized the binding of these PFO derivatives on murine macrophage-like cells whose cholesterol content was reduced or augmented. Our findings revealed that engineered PFO derivatives differentially associated with these cells in response to changes in cholesterol levels in the plasma membrane

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

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