Acidosis/GPR4 and Endothelial Cell Adhesion Figure 8. Activation of GPR4 by hypercapnic acidosis stimulates HUVEC adhesion molecule expression, cAMP production, and adhesiveness. (A–C) HUVEC/Vector, HUVEC/GPR4, or HUVEC/GPR4 R115A cells were treated with ambient air, 5% CO2, or 20% CO2-buffered EGM-2 pH media for 5 h. Total RNA was isolated and mRNA levels of E-selectin (A), VCAM-1 (B), and ICAM-1 (C) were determined by real-time RT-PCR. Values were normalized to the housekeeping gene GAPDH. The expression level of the target gene in HUVEC/Vector cells under ambient air (,pH 8.4) was set as 1. (D) The intracellular cAMP level in HUVEC/Vector and HUVEC/GPR4 cells treated with the CO2-buffered pH media was determined by the cAMP assay as described in the ‘‘Materials and Methods’’. *, P,0.05; **, P,0.01; compared with the 5% CO2 group. (E) HUVEC/Vector and HUVEC/ GPR4 cells were treated with the CO2-buffered pH media as indicated for 5 h. The cell adhesion assay was performed as described in the ‘‘Materials and Methods’’. ***, P,0.001; compared with the 5% CO2 group. All the above results are representative of at least two independent experiments. Error bars are the mean 6 SEM. doi:10.1371/journal.pone.0027586.g008 tumors in which inflammation plays an important role in disease progression. Interestingly, it has been shown that inflammatory stimuli TNF-a and H2O2 can induce GPR4 mRNA expression in human brain microvascular endothelial cells but not dermal microvascular endothelial cells [63]. These data suggest that the acidosis/GPR4 pathway may serve as a potential target for modulating vascular adhesion, endothelium-blood cell interaction, and inflammatory responses. Acidosis can modulate the expression of a wide range of genes in various cell systems such as renal, intestinal, and cancer cells [64,65,66]. Previous studies showed that acidosis is important for hypoxia/inflammatory stimuli-induced ICAM-1 expression in cultured endothelial cells [67]. Acidification of extracellular pH increases the expression of a key inflammatory enzyme cycloox- ygenase-2 (COX-2) in endothelial cells [68]. However, the pH- sensing mechanisms of endothelial cells in response to acidosis are PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27586