Effect of cell density on histone H3 acetylation at the E-selectin promoter.

<p>Schema of the E-selectin gene and the location of the primers used in the ChIP assay. Chromatin was prepared from sparse and confluent HUVECs monolayers treated with (+) or without (−) 1 ng/ml TNFα for 30 min, and the ChIP assay was performed using the anti-acetyl histone H3 (AcH3) antibody. Real-time PCR analysis of the ChIP samples was performed using primers designed to amplify the E-selectin promoter region (−195 to −67), which contains the cytokine response region (CRR) and the E-selectin extreme 3′ region (+13914 to +13977) (*<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments.</p

Effect of cell density on E-selectin protein expression levels in HUVECs.

<p>(<b>A</b>) HUVECs were cultured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090502#s2" target="_blank">Materials and Methods</a>. Photomicrographs were obtained using phase-contrast microscopy (20× magnification). (<b>B</b>) E-selectin protein expression levels under the sparse (S) and confluent (C) conditions. HUVECs cultured under both the conditions were treated with (+) or without (−) 1 ng/ml TNFα for 4 h, and total cell extracts were prepared. Equal amounts of protein were separated using SDS-PAGE, and western blot analysis was performed using the anti-E-selectin antibody (Santa Cruz Biotechnology). (<b>C</b>) Flow cytometric analysis of E-selectin cell surface expression. HUVECs were stimulated with or without 1 ng/ml TNFα for 4 h, and flow cytometry was performed using the anti-E-selectin antibody (clone 7A9) (*<i>p</i><0.05). The values are expressed as mean ± SD of three independent experiments.</p

Effect of cell density on TNFα-induced MAPK and NF-kB activation in HUVECs.

<p>Western blot of phosphorylated (p) and total JNK, p38 MAPK (<b>A</b>), and NF-κB p65 (<b>B</b>). HUVECs were stimulated with (+) or without (−) 1 ng/ml TNFα for 15 min. (<b>C</b>) p65 was detected using western blot analysis of the nuclear and cytoplasmic fractions prepared from HUVECs treated with or without 1ng/ml TNFα for 30 min. Representative western blots are shown.</p

Chromatin accessibility differed between sparse and confluent HUVEC monolayers.

<p>CHART-PCR of the E-selectin promoter region. The location of primers used in CHART-PCR assays is shown. Nuclei were isolated from sparse and confluent HUVECs monolayers treated with (+) or without (−) 1 ng/ml TNFα for 60 min. The isolated nuclei were incubated with 10 U of micrococcal nuclease for 10 min at room temperature. Genomic DNA was extracted and quantified using real-time PCR relative to DNA prepared from undigested nuclei (*<i>p</i><0.05, **<i>p</i><0.01). The values are expressed as mean ± SD of three independent experiments. CRR indicates cytokine response region.</p

Effect of cell density on E-selectin mRNA expression and mRNA stability in HUVECs.

<p>(<b>A</b>) Relative E-selectin mRNA levels measured using quantitative RT-PCR. RNA was isolated 2 h after stimulation with 1 ng/ml TNFα and 5 ng cDNA was used in each reaction (*<i>p</i><0.05 vs sparse). (<b>B</b>) E-selectin mRNA stability in TNFα-activated HUVECs. HUVECs were stimulated with 1 ng/ml TNFα for 2 h and treated with actinomycine D (5 µg/ml) for the indicated time periods. RNA was subjected to real-time RT-PCR. For each condition, E-selectin expression levels were normalized against those of GAPDH. The values were expressed proportional to that at baseline (time zero). The values are expressed as mean ± SD of three independent experiments.</p

7-Ketocholesterol increases the expression of adhesion molecules and cytokines in human umbilical vascular endothelial cells (HUVECs).

<p>(A) HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml tumor necrosis factor (TNF)-α for an additional 4 h. The levels of E-selectin, ICAM-1, and VCAM-1 protein expression were analyzed by western blotting. Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml TNF-α for an additional 2 h. IL-8 and MCP-1 mRNA levels were analyzed by RT-qPCR. Data are shown as mean ± standard errors of the means. *p < 0.05, **p < 0.01 by one-way analysis of variance followed by Tukey’s test.</p

Schematic representation of the signaling pathways involved in the 7-ketocholesterol-induced leukocyte-endothelial interactions.

<p>7-ketocholesterol induces E-selectin expression mediated by ATF-2 and involves the p38MAPK activation pathway, which together increase the number of leukocyte interaction to endothelial cells.</p

Effects of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced mitogen-activated protein kinase (MAPK) activity in human umbilical vascular endothelial cells (HUVECs).

<p>(A) HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol for each indicated time interval, followed by stimulation with 0.1 ng/ml TNF-α for an additional 15 min. Western blotting was used to evaluate the levels of p38, phospho-p38, JNK and phospho-JNK proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, followed by incubation with 5 μM p38MAPK inhibitor (SB203580) for 30 min and stimulation with TNF-α for an additional 4 h. A non-static adhesion assay was performed. Fluorescently labeled THP-1 cells were added to the HUVECs and allowed to adhere for 10 min under rotating conditions. Data are shown as means ± standard errors of the means (SEM). *p < 0.05 by a one-way analysis of variance followed by Tukey’s test. (C) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, incubated with 5 μM p38 MAPK inhibitor (SB203580) for 30 min and stimulated with TNF-α for an additional 4 h. Western blotting was used to evaluate the expression of E-selectin, ICAM-1, and VCAM-1 proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown.</p

THP-1 cell adhesion to HUVECs (Flow chamber assay) and expression of E-selectin in HUVECs.

<p>(A) SRPO significantly reduced the number of PRP-induced THP-1 cell that adhered to HUVECs. HUVEC monolayers were stimulated with 3 ng/ml TNF-α for 3.5 h and exposed to PPP or PRP for 20 min. PRP was pretreated or not with SRPO (10 μM) just before addition to the HUVECs. THP-1 cells were perfused over activated HUVEC monolayers at a flow rate of 1.0 dyne/cm². Adhesion assays were performed as described in Materials and Methods. Data are the mean ± SD of three independent experiments in each group. (B) SRPO significantly reduced the expression of PRP-induced E-selectin in HUVECs. E-selectin expression was determined by FIA as described in Materials and Methods. Data are the mean ± SD of 6 independent experiments in each group.</p

Effects of SRPO on HFFD-induced obesity.

<p>(A) Body weight (n = 17, 28, 29). (B) Epididymal fat weight (n = 13, 18, 18). (C) Liver weight (n = 17, 23, 25). Values are the mean ± SE in each group. (Abbreviations: NC = normal chow; HFFD = high-fat high-fructose diet; VEH = vehicle; SRPO = Sarpogrelate hydrochloride.)</p