Protocol details for the fluorescent immunohistological stainings (CD105 and CD45).

<p>Protocol details for the fluorescent immunohistological stainings (CD105 and CD45).</p

Overview of the experiments that were conducted in C57BL/6J <i>LDLR+/+</i> and <i>LDLR-/-</i> mice.

<p>Overview of the experiments that were conducted in C57BL/6J <i>LDLR+/+</i> and <i>LDLR-/-</i> mice.</p

The effect of clamping on CD105 presence, displayed as percentage fluorescence detected in intima and media.

<p>Results of wildtype and <i>LDLR</i> knockout mice are shown immediately after clamping (Median and IQR, 5–95 percentile). * p<0.05, *** p<0.0001.</p

Relative endothelium-dependent relaxation due to cumulative ACh administration after PE-induced preconstriction (Mean ± SD).

<p>Dose-response curves are shown for wildtype (A,B) and LDLR-/- (C,D) mice, both for segments tested immediately after clamping (A,C) or after one month survival (B,D). * p<0.05.</p

Atherosclerosis Alters Loading-Induced Arterial Damage: Implications for Robotic Surgery - Fig 2

<p>Representative images of osteopontin staining, showing the condition of the lowest score (A) and the highest score (B). *: atherosclerotic plaque, scale bar: 200μm.</p

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

<div><h3>Rationale</h3><p>Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.</p> <h3>Objectives</h3><p>We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.</p> <h3>Methods and Results</h3><p>HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL <em>in vitro</em> we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM <em>in vivo</em>. HDL also reversed the LDL-induced monocyte and granulocyte differentiation <em>in vitro</em>.</p> <h3>Conclusion</h3><p>Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.</p> </div

Hyercholesterolemia induced increased LSK percentage in PB and BM in LDLr^−/− mice.

<p>(A) Quantification of LSK cells in the PB of WT and LDLr^−/− mice two months after normal and high fat diet. n = 5–12. (B) The percentage of LSK cells in BM of WT and LDLr^−/− mice on normal and high fat diets. (C) Quantification of granulocyte/macrophage progenitors (GMP) in BM of WT and LDLr^−/− mice on normal and high fat diets.</p

LDL increased myeloid cell production in vitro.

<p>Sorted LSK cells of C57BL/6 mice were seeded at 1000 cells per well in 96-well plate and cultured in SFEM supplemented with IL-3, IL-6 and SCF for 14 days. LDL or LDL plus HDL were added as indicated. GM-CSF was used as the positive control. Total cells and cells with morphological features of differentiated cells were enumerated under the microscopy. (A) Data were expressed as the percentage of undifferentiated cells in total cells. Cells were stained with antibodies against CD11b PE and Ly-6c PE-Cy7 (B), CD11b PE and F4/80 APC-Cy7 (C), and CD11b PE and Ly-6G APC (D) for FACS analysis.</p

SR-BI expression on HSPC and the effect of apoA-I infusion on HSPC in PB and BM.

<p>(A) TBMCs were stained with rabbit anti-mouse SR-BI and then goat anti-rabbit Alexa 488, followed by LSK staining. SR-BI expressing LSK cells were quantified by FACS based on isotype control stains. Data are expressed as the percentage of SR-BI+ LSK cells in LSK cell population. (B) The percentage of LSK cells in BM of mice infused with saline vs. purified human apoA-I 8 mg/kg every 2 days for 3 injections.</p

LDL modulates Lin- cell motility which is partially mediated via pERK.

<p>To study the effect of LDL on HSPC function, Lin- cells of CD45.2 mice were exposed to 0 or 100 µg/ml LDL in the presence or absence of pERK inhibitor U0126 for 24 hours and then subjected for adhesion and migration assays. (A) Cells were seeded onto fibronectin-coated plates for 4 hours. After extensive washing, adhered cells were stained with crystal violet and counted under the microscopy. (B) Cells were loaded into transwells and allowed to migrate for 4 hours. Cells in the upper chamber of the transwells were removed by swaps. Cells migrated to the lower surface of the transwells were stained with Hoechst. Adherent cells or migrated cells were numerated under the microscope, averaging the number of cells in 5 random fields.</p