Anti-ICAM-1 stimulation induces an increase in PKCα^{Thr 638} phosphorylation through XO/PLC/ERK1/2 activities but not by oxidation. A,B,D

<p>) 70% confluent monolayers of HMVECLs were pretreated with TNFα to induce ICAM-1 expression. Then, the endothelial cells were were treated for 1 hr with the pharmacological inhibitors allopurinol (0.3 mg/ml), PD98056 (20 µM), U73122 (10 µM), apocynin (4 mM), Ly294002 (100 nM), Go6976 (2.3 nM), PP2 (10 µM), apocynin (4 mM) or the solvent control DMSO (0.01%). The cells were stimulated with anti-ICAM-1-coated beads and examined for (<b>A,B</b>) PKCα^Thr638 phosphorylation and total PKCα by western blot or examined for (<b>D</b>) cytotoxicity by the Vybrant cytotoxicity assay. In panel D, 200 µM H₂O₂ was used as a positive control for cytotoxicity. <b>C</b>) To label non-oxidized cysteines, BIAM was added to the cell lysates. PKCα was immunoprecipitated and BIAM labeling was detected by western blot with HRP-conjugated streptavidin. The blots were reprobed for total PKCα. The positive control for oxidation includes lysates oxidized with 200 µM H₂O₂ for 20 min before addition of BIAM. Loss of BIAM labeling in the western blot indicates PKCα oxidation. Shown are the means ± SEM from 3 experiments. *, p<0.05 compared to nonstimulated (NS) controls.</p

Inhibition of endothelial cell ERK1/2 with an ERK2 dominant negative plasmid (K52R) blocks VCAM-1-dependent leukocyte migration.

<p>Two million mHEVa endothelial cells, that were grown to 70∼80% confluence, were suspended with trypsin and transfected with GFP-ERK2 K52R or vector control using the Amaxa nucleofector method. Transfected cells were seeded onto 9 cm² culture slides. Four hours after nucleofection, cells were examined for ERK1/2 expression or used in TEM assays. Greater than 60% of the endothelial cells were transfected as determined by flow cytometry with detection of GFP-ERK2 K52R (data not shown). The transfected endothelial cells formed confluent monolayers in 4 hours and were greater than 85% viable (data not shown). In addition, to block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. <b>A</b>) The endothelial cells were washed with ice-cold phosphate-buffered saline and examined by western blot for ERK1/2 and β-actin expression. Lanes 1 and 2 are two samples of vector-treated cells. Lanes 3 and 4 are two samples of cells treated with GFP-ERK2 K52R. <b>B</b>) Leukocyte TEM under laminar flow at 2 dynes/cm², <b>C</b>) Leukocyte association assay under laminar flow at 2 dynes/cm². Data for each panel are from 3 experiments. *, p<0.05 compared to non-treated (NT) or vector transfected groups.</p

Mechanisms for VCAM-1 activation of ERK1/2 in HMVEC-L.

<p>Treatment of HMVEC-L cells overnight with 10ng/ml TNF-α induced VCAM-1 expression (data not shown). <b>A</b>) Confluent monolayers of TNF-α-treated HMVEC-Ls were nontreated (NT) or treated with 27 µg/ml anti-VCAM-1 plus 15 µg/ml of a secondary antibody to crosslink and stimulate VCAM-1. Phosphorylation of ERK1/2 Thr202/Tyr204 (P-ERK1/2) and total expression of ERK1/2 was examined by western blot using rabbit anti-phospho ERK1/2 Thr202/Tyr204 (1/1000) followed by HRP-conjugated anti-rabbit (1/2000) and ECL detection. <b>B</b>) Confluent monolayers of TNF-α stimulated HMVEC-L cells in 12 well plates were nontreated or incubated for 30 minutes with the solvent control DMSO, apocynin (4 mM), Gö-6976 (2.3 nM) or CinnGEL 2-methylester (10 µM). These endothelial cells were then stimulated with anti-VCAM-1 antibody plus a secondary antibody for 15 minutes. The apocynin, DMSO, Gö-6976 or CinnGEL 2-methylester had no effect on endothelial cell viability as determined by trypan blue exclusion and had no effect on VCAM-1 expression as determined by flow cytometry (data not shown). Representative western blots are shown. Data presented are the mean ± standard deviation from 3 experiments. The phosphorylation status of ERK1/2 is presented as the fold increase in the ratio of the relative intensity of P-ERK1/2 divided by the relative intensity of the loading control (total ERK1/2). *, p<0.05 compared to <b>A</b>) NT cells or <b>B</b>) anti-VCAM-1 stimulated cells.</p

Uptake of tocopherols by HMVECLs.

<p>At 70% confluence, HMVECLs cells were stimulated for 6 hrs with 10 ng/ml TNFα and then were treated for 16 hrs with a dose curve of α-tocopherol (α-toc) and/or γ-tocopherol (γ-toc) or with the vehicle control (DMSO 0.01%). Cells were washed, cell pellet weighed and tocopherol uptake was measured by HPLC/ECD. Data are expressed as µg tocopherol per g cells. Shown is the mean± SEM n = 3–5.</p>*<p>p<0.05 compared to the DMSO control.</p

ICAM-1 activates PKCα but not PKCβII in HMVECLs. A

<p>) At 70% confluence, HMVECLs cells pretreated with 10 ng/ml TNFα to induce ICAM-1 expression. At 24 hrs, the cells were suspended and immunolabeled with anti-ICAM-1 antibodies and examined by flow cytometry for ICAM-1 expression. <b>B–D</b>) HMVECLs were pretreated with TNFα as in panel A. At 24 hrs, the endothelial cells were nonstimulated (NS) or stimulated with a confluent monolayer of anti-ICAM-1-coated beads or the control anti-PECAM-1-coated beads for 20 minutes in <b>B</b> and <b>D</b> or for the times indicated in <b>C</b>. The cells were lysed and the activation of PKCβII and PKCα was examined by western blot with <b>B</b>) anti-phospho-PKCβII^Thr641 or anti-PKCβII or <b>C–D</b>) anti-phospho- PKCα^Thr638 or anti-PKCα. Shown are representative blots. Shown are the mean ± SEM of 3 experiments. NT, nontreated. *, p<0.05 as compared to the nontreated (NT) groups.</p

D-α-tocopherol inhibits ICAM-1-activated PKCα but not ERK1/2 in HMVECLs and the inhibition by d-α-tocopherol is abrogated by d-γ-tocopherol.

<p>At 70% confluence, HMVECLs cells were pretreated with TNFα for 6hrs and then treated with tocopherols or the solvent control 0.01% DMSO for 16 hrs. <b>A</b>) d-α-tocopherol (α-toc) regulation of ICAM-1-stimulated PKCα Thr⁶³⁸ phosphorylation (PKCα P-Thr⁶³⁸). <b>B</b>) d-γ-tocopherol (γ-toc) regulation of ICAM-1-stimulated PKCα P-Thr⁶³⁸. <b>C</b>) d-α-tocopherol + d-γ-tocopherol regulation of ICAM-1-stimulated PKCα P-Thr⁶³⁸. <b>D</b>) d-α-tocopherol does not alter background PKCα P-Thr⁶³⁸ (no anti-ICAM-1). <b>E</b>) d-α-tocopherol does not regulate ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation (P-ERK1/2). <b>F</b>) d-γ-tocopherol does not regulate ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation. Shown are the means ± SEM from 3 experiments. *, p<0.05 as compared with the anti-ICAM-1-stimulated groups.</p

Inhibition of MEK, which is known to activate ERK1/2, blocks VCAM-1-dependent spleen leukocyte TEM.

<p>Confluent monolayers of mHEVa cells were nontreated (NT) or treated for 30 minutes with the MEK inhibitors PD98059 (20 µM) or U0126 (20–40 µM). To block leukocyte binding to VCAM-1, the endothelial cells were treated with a blocking anti-VCAM-1 antibody without a secondary crosslinking antibody. <b>A–D</b>) Splenic leukocytes were added to the endothelial monolayer, allowed to briefly settle to mediate cell contact and then exposed to 2 dynes/cm² laminar flow for 15 minutes to examine migration (<b>A and B</b>) or 2 minutes to examine leukocyte-endothelial cell association (<b>C and D</b>). Then, cells were washed and fixed in 3% paraformaldehyde for 1 hour and examined by phase contrast microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Ager1" target="_blank">[65]</a>. Non-migrated leukocytes are phase-light and migrated leukocytes appear as phase-dark <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Ager1" target="_blank">[65]</a>. We previously reported that the leukocytes that migrated are >88% lymphocytes as determined by flow cytometry <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Tudor1" target="_blank">[6]</a>. <b>E</b>) Relative cytotoxicity was determined by the G6PDH assay; PD98059 (20 µM) and U0126 (40 µM) were not cytotoxic as compared to the nontreated control cells. Data in each panel are from 3 experiments. *, p<0.05 compared to <b>A,C</b>) NT groups, <b>B,D</b>) 0 minutes groups, or compared to DMSO-treated or last washes (data not shown). Inhibitors had no effect on cell viability, as determined by Trypan blue exclusion (data not shown).</p

ICAM-1 activation of PKCα phosphorylation in HMVECLs is mediated by xanthine oxidase stimulation of ERK1/2.

<p>70% confluent monolayers of HMVECLs were pretreated with TNFα to induce ICAM-1 expression. <b>A</b>) Time course for anti-ICAM-1-coated bead stimulation of ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation (P-ERK1/2). *, p<0.05 as compared to the nontreated control group. <b>B</b>) TNFα-pretreated HMVECLs were incubated for 1 hr with the pharmacological inhibitors allopurinol (0.3 mg/ml), U73122 (10 µM), and Go6976 (2.3 nM), apocynin (4 mM), or the solvent control DMSO (0.01%). The cells were nonstimulated (NS) or stimulated with anti-ICAM-1-coated beads and examined by western blot for ERK1/2 Thr²⁰²/Tyr²⁰⁴ phosphorylation and total ERK1/2. Shown are the means ± SEM from 3 experiments. Panel A: *, p<0.05 as compared to 0 minutes. Panel B: *, p<0.05 as compared to the anti-ICAM-1-stimulated group.</p

VCAM-1 induces an increase in phosphorylation of ERK1/2 in endothelial cell lines under static and laminar flow conditions.

<p>Confluent monolayers of mHEVa cells were stimulated with 27 µg/ml anti-VCAM-1 (or the binding control anti-CD98) plus 15 µg/ml of a secondary antibody. ERK1/2 Thr202/Tyr204 phosphorylation (P-ERK1/2) and total ERK1/2 was determined by western blot. <b>A</b>) Time course for anti-VCAM-1 activation of ERK1/2 Thr202/Tyr204 phosphorylation under static conditions. <b>B</b>) Stimulation of VCAM-1 for 30 minutes and time course for antibody crosslinking of the control CD98 under static conditions. <b>C</b>) Confluent monolayers of HEV were non-treated (NT) or stimulated with anti-VCAM-1 or the control anti-CD98 under 2 dynes/cm² laminar flow or nontreated under static conditions for 15 minutes. *, p<0.05 compared to <b>A</b>) 0 minutes, <b>B</b>) NT and <b>C</b>) the NT,flow group. In panel <b>C</b>) **, p<0.5 compared to the anti-VCAM-1, flow group.</p

Mechanisms for VCAM-1 activation of ERK1/2 in endothelial cell lines.

<p>Monolayers of mHEVa cells were nontreated (NT) or incubated for 30 minutes with apocynin (4 mM), PD98059 (30uM), U0126 (40 µM), Gö-6976 (2.3 nM), CinnGEL 2-methylester (10 µM), or catalase (5000 U/ml) where indicated. These are the optimal doses for these inhibitors as we have previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Matheny1" target="_blank">[5]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-Deem1" target="_blank">[7]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026706#pone.0026706-AbdalaValencia1" target="_blank">[8]</a>. At these concentrations, none of the inhibitors had any significant effects on the basal level of ERK1/2 in the absence of anti-VCAM-1 stimulation (data not shown). After treatement with the inhibitor, the endothelial cells were stimulated with anti-VCAM-1 antibody plus a secondary antibody to crosslink VCAM-1 for 30 min under static conditions. We examined phosphorylation of <b>A,B,F</b>) ERK1/2 Thr202/Tyr204 (P-ERK1/2), <b>C</b>) PKCα Thr638 (P-PKCα), or <b>D</b>) MEK1/2 Ser217/221 (P-MEK1/2) by western blot. <b>E</b>) mHEVa cells were treated with exogenous 1 µM H₂O₂ for 10–60 minutes and ERK1/2 Ser217/221 phosphorylation was determined by western blot. The phosphorylation status of ERK1/2 Thr202/Tyr204, PKCα Thr638, or MEK1/2 Ser217/221 is presented as the fold increase in the ratio of the relative intensity of the phosphorylated enzyme to total ERK1/2, total PKCα or total MEK1/2 expression. Representative western blots are shown and data are presented as the mean ± standard deviation from 3 experiments. (<b>A</b>, <b>B</b>) *, p<0.05 less than anti-VCAM-1-treated group. (<b>C–F</b>) *, p<0.05 greater than NT.</p