Potential molecular mechanisms (hypothesis) of resistin-induced endothelial permeability.

<p>Resistin may interact with toll-like receptor 4 (TLR4), inducing a TLR4-mediated signaling cascade such as the activation of p38, NADPH oxidase and transcriptional factor CREB, which directly and/or indirectly reduces the expression of ZO-1 and occludin at transcriptional and/or post-transcriptional levels.</p

Effect of resistin on the permeability of HCAECs.

<p>Endothelial monolayer permeability was measured using the Costar Transwell permeability system with a fluorescence-labeled dextran tracer. HCAECs were treated with different concentrations of resistin (40 and 80 ng/mL), with or without Ginkgolide A (GA, 5 µM) pre-treatment, for 24 hours. Cells treated with TNF-α (2 ng/mL) served as a positive control. The results of the resistin-treated cells were compared with the results of control cells (n = 3, *<i>P</i><0.05). The results of the cells that were pretreated with Ginkgolide A for 30 minutes before incubating them with resistin for 24 hours, were compared with results of the resistin-treated cells (n = 3, ^#<i>P</i><0.05). The experiment was repeated thrice.</p

Effect of resistin on ROS production in HCAECs.

<p>Superoxide anion production was analyzed by DHE staining and flow cytometry analysis. (<b>A</b>)<b>.</b> Histogram showing DHE positively stained cells. (<b>a</b>). Normal HCAECs stained with DHE served as staining control. (<b>b, c,</b> and <b>d</b>) HCAECs were treated with different concentrations of resistin (40 and 80 ng/mL), or pretreated with Ginkgolide A and then incubated with resistin (80 ng/mL) for 24 hours, before staining with DHE (3 µM, 20 minutes). (<b>B</b>)<b>.</b> Bar diagram showing the average percentage of positively stained cells from three separate experiments. The results of the resistin-treated cells were compared with the results of control cells (n = 3, *<i>P</i><0.05). The results of the cells pretreated with Ginkgolide A for 30 minutes followed by resistin treatment for 24 hours, were compared with the results of resistin-treated cells (n = 3, ^#<i>P</i><0.05).</p

Effects of MAPK inhibitors on resistin-induced permeability in HCAECs.

<p>The effect of MAPK inhibitors on resistin-induced permeability in HCAECs was determined using a Transwell permeability assay. HCAECs were treated with resistin (80 ng/mL) alone, or pre-treated with each specific inhibitor of MAPKs (SB203580 for p38, SP600125 for JNK, and PD98059 for EEK1/2) for 30 minutes. Cells treated with TNF-α served as a positive control. The results of the resistin-treated cells were compared with the results of control cells (n = 3, *<i>P</i><0.05). The results of the cells pretreated with MAPK inhibitor for 30 minutes, followed by resistin treatment for 24 hours,were compared with the results of the resistin-treated cells (n = 3, ^#<i>P</i><0.05).</p

Effect of resistin on phosphorylation of MAPKs in HCAECs.

<p>(<b>A</b>)<b>.</b> The activation of MAPKs (ERK1/2, JNK, and p38) was determined using the Bio-Plex luminescence assay in HCAECs treated with resistin (80 µg/mL) for different time periods (0, 5, 10, 20, 30, 45, 60, and 90 minutes). The phosphorylated and the total protein for each MAPK were determined. (<b>B</b>)<b>.</b> HCAECs were incubated with or without resistin, or pretreated with MnTBAP before treating them with resistin (80 ng/mL) for 45 minutes. The protein levels of MAPK (total and phosphorylated proteins) were determined by Western Blot. β-actin was used as a loading control.</p

Effects of resistin on mRNA and protein levels of junctional molecules in HCAECs.

<p>(<b>A</b>)<b>.</b> HCAECs were treated with resistin (20, 40, and 80 ng/mL) for 24 hours, or pretreated with Ginkgolide A (5 µM) for 30 minutes before resistin treatment (40 ng/mL) for 24 hours. The mRNA levels of junction molecules (VE-cadherin, ZO-1, and occluding) were determined by real time PCR. The relative mRNA levels of each gene were normalized to the expression of a house keeping gene β-actin. The results of the resistin-treated cells were compared with the results of the control cells (n = 3, *<i>P</i><0.05). The results of the cells that were pretreated with Ginkgolide A for 30 minutes and then treated with resistin (40 ng/mL) for 24 hours were compared with results of the resistin-treated cells (n = 3, ^#<i>P</i><0.05). (<b>B</b>)<b>.</b> The protein levels of VE cadherin, ZO-1, and occludin were determined by Western blot analysis after resistin treatment and compared with controls. Equal loading control was monitored by reprobing the blot with anti-β-actin antibody. Western blot band density ratio for each tight junction protein and control β-actin was measured with ImageJ (1.47) software (NIH). To determine the effect of adding an antioxidant, cells were pretreated with MnTBAP (2 µM) for 30 minutes, and then incubated with resistin.</p

Flow cytometry analysis of junctional molecules in HCAECs.

<p>HCAECs were pretreated with Ginkgolide A (GA) for 30 minutes (or left untreated), and then treated with resistin (80 ng/mL) for 24 hours. The protein levels of junction molecules were determined by flow cytometry analysis. (<b>A</b>)<b>.</b> These representative histograms show the percentage of positively stained cells for each specific antibody against each junction protein, including VE-cadherin, ZO-1, and occludin. (<b>B</b>)<b>.</b> Bar diagram showing the average percentage of positively stained cells in three separate experiments. The results of the resistin-treated cells were compared with results of control cells (n = 3, *<i>P</i><0.05).</p