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. 2013 Sep;3(3):578–588. doi: 10.1086/674337

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© 2013 by the Pulmonary Vascular Research Institute. All rights reserved.

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Toll-like receptor 4 (TLR4) expression in pulmonary artery endothelial cells (PAECs) preconditioned in hypoxia and treated with lipopolysaccharide (LPS) and TAK-242. Preliminary experiments showed that LPS-stimulated expression of TLR4 messenger RNA (mRNA) peaked 4 hours after exposure followed by a decrease to basal levels at 24 hours (not shown). Accordingly, PAECs were incubated in conditions of hypoxia or normoxia for 24 hours followed by treatment with LPS or vehicle for an additional 4 hours, and then TLR4 mRNA was measured ( for all groups). LPS increases the expression of TLR4 severalfold compared with untreated cells (, bars 1 vs. 2). A t test of TLR4 mRNA from cells treated with hypoxia alone compared with normoxia (bars 1 vs. 5) revealed a hypoxia-induced decrease in TLR4 (). Preconditioning of the cells in hypoxia prior to treatment with LPS leads to a reduction in TLR4 expression (, bars 2 vs. 6). The effect of TAK-242 on LPS-stimulated TLR4 mRNA expression was determined by incubating PAECs in hypoxic or normoxic conditions for 24 hours with TAK-242 or vehicle followed by treatment with LPS or vehicle for an additional 4 hours ( separate isolates of PAECs). TAK-242 decreased the expression of LPS-stimulated TLR4 cells incubated in both normoxic and hypoxic conditions (, analysis of variance [ANOVA]; other pairwise comparisons are as per the graph).

Inline graphic — Toll-like receptor 4 (TLR4) expression in pulmonary artery endothelial cells (PAECs) preconditioned in hypoxia and treated with lipopolysaccharide (LPS) and TAK-242. Preliminary experiments showed that LPS-stimulated expression of TLR4 messenger RNA (mRNA) peaked 4 hours after exposure followed by a decrease to basal levels at 24 hours (not shown). Accordingly, PAECs were incubated in conditions of hypoxia or normoxia for 24 hours followed by treatment with LPS or vehicle for an additional 4 hours, and then TLR4 mRNA was measured ( for all groups). LPS increases the expression of TLR4 severalfold compared with untreated cells (, bars 1 vs. 2). A t test of TLR4 mRNA from cells treated with hypoxia alone compared with normoxia (bars 1 vs. 5) revealed a hypoxia-induced decrease in TLR4 (). Preconditioning of the cells in hypoxia prior to treatment with LPS leads to a reduction in TLR4 expression (, bars 2 vs. 6). The effect of TAK-242 on LPS-stimulated TLR4 mRNA expression was determined by incubating PAECs in hypoxic or normoxic conditions for 24 hours with TAK-242 or vehicle followed by treatment with LPS or vehicle for an additional 4 hours ( separate isolates of PAECs). TAK-242 decreased the expression of LPS-stimulated TLR4 cells incubated in both normoxic and hypoxic conditions (, analysis of variance [ANOVA]; other pairwise comparisons are as per the graph).