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

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