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 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

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

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

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

Continuous real-time recordings of adhesion probability of VSMC to COL-I coated AFM probe following ADO or NO administration.

<p><b>(A)</b> A representative individual experiment shows the decrease in the adhesion probability after introduction of ADO in the cell bath. <b>(B)</b> Decrease in the group average adhesion probability after addition of ADO in the cell bath (n = 10). <b>(C)</b> Average adhesion probability showed a significant decrease in adhesion probability following addition of ADO. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> NO donor dramatically reduced adhesion probability as shown in a representative individual example. <b>(E)</b> Average adhesion events decreased after treatment with NO (n = 10). <b>(F)</b> Average adhesion probability shows a significant decrease in adhesion probability after the addition of NO donor to the cell bath. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>).</p

Continuous real-time VSMC E-modulus recordings following ADO or NO.

<p><b>(A)</b> VSMC E-modulus immediately reduced after addition of ADO (10⁻⁴ M) as shown in a representative example. <b>(B)</b> Group average of VSMC E-modulus after addition of ADO (n = 10) in cell bath. <b>(C)</b> Average E-modulus summed across all time points for the group of VSMCs before and after ADO (n = 10, *<i>P<0.05</i>). <b>(D)</b> A representative single cell measurement shows decrease in VSMC E-modulus following addition of NO to the cell bath. <b>(E)</b> Alteration in group average of VSMC E-modulus before and after addition of NO (n = 10). <b>(F)</b> Average E-modulus summed for all time points before and after addition of NO showed a significant decrease in VSMC E-modulus (n = 10, *<i>P<0.05</i>). Data were presented as mean ± SEM.</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe following stimulation with ANG II.

<p>The adhesion probability is presented as the number of adhesion events per AFM retraction curve. <b>(A)</b> A representative individual example shows the increase in VSMC adhesion probability after stimulation with ANG II. <b>(B)</b> Average adhesion probability before and after addition of ANG II for experimental group (n = 10). <b>(C)</b> Average adhesion probability significantly increased after ANG II treatment. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>). <b>(D)</b> A representative single cell record of VSMC E-modulus shows the immediate increase in cell E-modulus after the addition of ANG II in cell bath (10⁻⁶ M). <b>(E)</b> Alteration in group average of VSMC E-modulus before and after stimulation with ANG II (n = 10). <b>(F)</b> Average E-modulus summed across all time points for the group significantly increased after addition of ANG II. Data were summed over 1800 s and were presented as mean ± SEM (n = 10, *<i>P<0.05</i>.</p

Continuous real-time recordings of E-modulus and adhesion probability of VSMC to COL-I coated AFM probe in sham control experiments.

<p><b>(A)</b> A representative individual example shows no effect on adhesion probability after addition of vehicle buffer. <b>(B)</b> No change in adhesion probability was shown in the group average adhesion probability before and after addition of vehicle buffer (n = 10). <b>(C)</b> Average adhesion probability summed across all time points for the group of VSMCs before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). <b>(D)</b> A representative single cell measurement shows no changes in E-modulus before and after addition of vehicle buffer in cell bath. <b>(E)</b> Group average VSMC E-modulus did not change before and after addition of vehicle buffer in cell bath (n = 10). <b>(F)</b> No significant difference was shown in the average E-modulus summed for all time points before and after addition of vehicle buffer (n = 10, <i>P>0.05</i>). Data were presented as mean ± SEM.</p

Representative force curve recorded by AFM.

<p><b>(A)</b> An example of an approach curve recorded by AFM (red). The blue diamond in the black square is the estimated contact point with the VSMC where the AFM cantilever is in contact with the cell plasma membrane and begins to bend. The blue line is the Hertz fitting to the approach curve. <b>(B)</b> Representative retraction force curve recorded by AFM for pre-drug period (control). <b>(C)</b> Representative retraction force curves after treatment with NO. <b>(D)</b> Representative retraction force curve after treatment with ANG II. The height between the paired red spots was used to compute the adhesion force and the rupture number was used to evaluate the adhesion probability. The 1.5 fold of the average noise (signal fluctuation) was set as the threshold of adhesion force and only the rupture force higher than threshold was considered as the real unbinding force between cell membrane and AFM cantilever. The rupture indicated by two green spots was considered as noise and omitted in adhesion probability computation (<b>C)</b>. The snaps with low gap height were not the particular characteristics of the NO or ADO treated force curve, they also appeared in control and ANG II treated force curve and were omitted as well.</p