Effects of Aβ, histamine, and lovastatin on P-selectin expression at the CEC surface.

<p>(<i>A</i>) Fluorescent micrographs of fluorescently-labeled P-selectin at the bEnd3 cells. (<i>B</i>) Relative P-selectin intensity at the bEnd3 cell surface and (<i>C</i>) the human primary CEC surface. ***p≤0.001, **p≤0.01 compare to the control; °°° p≤0.001 compare to the Aβ (1 µM) treatment group.</p

Effects of Aβ, and histamine on actin polymerization in CECs.

<p>(<i>A</i>) Fluorescent micrographs of Oregon-green phalloidin-labeled F-actin in bEnd3 cells. (<i>B</i>) Relative F-actin intensity in bEnd3 cells and (<i>C</i>) primary human CECs. ***p≤0.001, ** p≤0.01, * p≤0.05 compare to the control; °°° p≤0.001 compare to the Aβ (1 µM) treatment group.</p

Effects of Aβ, histamine, and lovastatin on adhesion probability at the bEND3 cell surface with AFM cantilever tips biofunctionalized by conjugating sLe^x at their surface.

<p>(<i>A</i>) Adhesion probability was measured for cells treated with histamine, Aβ, lovastatin and Aβ and lovastatin alone. Adhesion probability was calculated by normalizing the number of force curves with adhesion events by the total number of force curves. ***p≤0.001, **p≤0.01 compare to the control; °°°p≤0.001 compare to the Aβ (1 µM) treatment group. (<i>B</i>) bEND3 was treated with histamine, and adhesion probability was measured. A highest adhesion probability was obtained for the cantilever coated with sLe^x; whereas lower adhesion probabilities were obtained for the cantilever coated with biotin only, and cells treated with antibody of P-selectin, indicating that <i>F_mtf</i> measured in this study are highly molecularly specific through sLe^x-P-selectin bonding. ***p≤0.001compare to the sLe^x coating group.</p

Schematic descriptions of membrane tether formation and biofunctionalization for the AFM cantilever tip.

<p>Membrane tether formation mediated by sLe^x-selectin bonding during a monocyte rolling on the endothelial layer (<i>upper</i>) and the strategy using AFM cantilever tips bio-functionalized by sLe^x to characterize the mechanics of membrane tether adhesion (<i>lower</i>). (modified from Yves F. Dufrêne, 2008).</p

Membrane stiffness.

<p>(<i>A</i>) Membrane stiffness for cells treated with histamine, Aβ, lovastatin and Aβ, lovastatin alone, and latrunculin A. (<i>B</i>) Membrane stiffness is measured by calculating the slope (denoted by the arrow) from 5 nm indentation at the cell surface. ***p≤0.001, **p≤0.01 compare to the control; °°p≤0.01, °°°p≤0.001 compare to the Aβ (1 µM) treatment group.</p

TNFα alters occludin and cerebral endothelial permeability: Role of p38MAPK

<div><p>Occludin is a key tight junction (TJ) protein in cerebral endothelial cells (CECs) playing an important role in modulating blood-brain barrier (BBB) functions. This protein (65kDa) has been shown to engage in many signaling pathways and phosphorylation by both tyrosine and threonine kinases. Despite yet unknown mechanisms, pro-inflammatory cytokines and endotoxin (lipopolysaccharides, LPS) may alter TJ proteins in CECs and BBB functions. Here we demonstrate the responses of occludin in an immortalized human cerebral endothelial cell line (hCMEC/D3) to stimulation by TNFα (10 ng/mL), IL-1β (10 ng/mL) and LPS (100 ng/mL). Exposing cells to TNFα resulted in a rapid and transient upward band-shift of occludin, suggesting of an increase in phosphorylation. Exposure to IL-1β produced significantly smaller effects and LPS produced almost no effects on occludin band-shift. TNFα also caused transient stimulation of p38MAPK and ERK1/2 in hCMEC/D3 cells, and the occludin band-shift induced by TNFα was suppressed by SB202190, an inhibitor for p38MAPK, and partly by U0126, the MEK1/2-ERK1/2 inhibitor. Cells treated with TNFα and IL-1β but not LPS for 24 h resulted in a significant (p < 0.001) decrease in the expression of occludin, and the decrease could be partially blocked by SB202190, the inhibitor for p38MAPK. Treatment with TNFα also altered cell morphology and enhanced permeability of the CEC layer as measured by the FITC-dextran assay and the trans-endothelial electrical resistances (TEER). However, treatment with SB202190 alone could not effectively reverse the TNFα -induced morphology changes or the enhanced permeability changes. These results suggest that despite effects of TNFα on p38MAPK-mediated occludin phosphorylation and expression, these changes are not sufficient to avert the TNFα-induced alterations on cell morphology and permeability.</p></div

TNFα-mediated occludin band-shift and phosphorylation of ERK1/2 and p38MAPK in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (10 ng/mL) at 5, 10, 15, 30, 60 min (A). Cell lysates were collected and occludin, P- ERK, T-ERK, P-P38, T-P38 and β-actin expression pattern was analyzed by immunoblotting assay. Quantification of the proportion of occludin upper band were determined through PI_upper/PI_total/PI_β-actin and then normalized to control (B). phospho-ERK1/2 (C) and phospho-p38MAPK (D) were similarly quantified and plotted. Results are mean ± SD from 3 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (**P<0.01, ***P<0.001 compared with no treatment control).</p

Effects of TNFα, IL-1β and LPS on occludin band-shift in hCMEC/D3 cells.

<p>Cells were treated with or without TNFα (10 ng/mL) (A), IL-1β (10 ng/mL)(B) and LPS (100 ng/mL) (C) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and occludin and β-actin expression patterns were analyzed by immunoblotting assay as described in text. The density of upper band versus total upper and lower and normalized with β-actin was determined by measuring protein intensity as PIupper/PItotal/PIβ-actin. Results are mean ± SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (***P<0.001 compared with no treatment control).</p

Effects of TNFα on tyrosine and threonine phosphorylation of occludin.

<p>hCMEC/D3 cells were treated with or without 10 ng/mL TNFα for 15 min. Cell lysates were mixed with anti-tyrosine or anti-threonine antibody conjugated with protein A beads and then phosphorylated proteins in cell lysates were pulled down. Occludin and β-actin expression pattern was analyzed by immunoblotting assay.</p

Schematic description of transendothelial permeability assays.

<p>(A) Diagram depicting method for Dextran assay protocol. (B) Diagram depicting the measurement using the TEER protocol.</p