Acidosis/GPR4 and Endothelial Cell Adhesion Activation of GPR4 by acidosis increases the expression of endothelial adhesion molecules E-selectin, VCAM-1, and ICAM-1 During leukocyte extravasation, the early steps like leukocyte tethering, rolling and firm adhesion are associated with elevated expression of endothelial cell adhesion molecules, such as E- selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1), three extensively characterized endothelial adhesion molecules [53,54]. To inves- tigate the gene expression levels of these adhesion molecules, we used real-time RT-PCR to examine their mRNA levels in pH- treated HUVEC cells transduced with different GPR4 constructs. Levels of VCAM-1 and E-selectin mRNAs were increased by about 7-fold upon acidosis treatment in HUVEC/Vector cells expressing the endogenous level of GPR4. Moreover, overexpres- sion of GPR4 in synergy with acidosis further enhanced the gene expression of VCAM-1 and E-selectin substantially (Figs. 5A and B). Acidosis/GPR4 stimulated the expression of ICAM-1, however, to a lesser extent as there was both a minor increase in HUVEC/Vector cells and an increase of 10-fold in HUVEC/ Figure 6. Immunofluorescence staining of E-selectin, VCAM-1 and ICAM-1 in HUVEC cells. After treated with EGM-2/HEM media at pH 8.4, 7.4, or 6.4 for 5 h, HUVEC/Vector and HUVEC/GPR4 cells were fixed with 4% paraformaldehyde, incubated with E-selectin, VCAM-1 or ICAM-1 primary antibody, Rhodamine Red-conjugated secondary antibody, and then detected under a fluorescence microscope (106 objective). For an accurate comparison of fluorescence signals, each group of images (e.g. E-selectin/Vectors cells, pH 8.4, pH 7.4, and pH 6.4) was taken with the same exposure time. The results are representative of three independent experiments. doi:10.1371/journal.pone.0027586.g006 PLoS ONE | www.plosone.org 9 November 2011 | Volume 6 | Issue 11 | e27586