Acidosis/GPR4 and Endothelial Cell Adhesion transduced with those two miRNAs that inhibited the endog- enous GPR4 expression (Fig. 1E). These results suggest that the expression of GPR4 is required for acidosis-stimulated HUEVC adhesion. We next examined the time course of acidosis/GPR4- induced endothelial cell adhesion. HUVEC/GPR4 cells were treated with EGM-2/HEM media at pH 6.4, 7.4 and 8.4 for 1, 3, 5, 24, and 48 hours and then U937 monocytes were added for the adhesion assay. As shown in Figure 1F, by 1 hour we did not observe an increase of HUVEC cell adhesion. By + + 3 hours, pH 6.4 (400 nM H ), compared to pH 8.4 (4 nM H ) and pH 7.4 (40 nM H+), significantly augmented the adhe- siveness of HUVECs. By 5 to 24 hours, we observed the peak induction of HUVEC adhesion by acidic pH. By 48 hours, the induction was decreased when compared to that at 5 and 24 hours. The results suggest that the acidosis/GPR4-induced HUVEC adhesion occurs within 3 hours after the acidic pH stimulation. Figure 3. Involvement of cAMP in acidosis/GPR4-induced HUVEC adhesion. (A) The intracellular cAMP level in pH-treated HUVEC/Vector cells was determined by the cAMP assay as described in the ‘‘Materials and Methods’’. *, P,0.05; compared with pH 8.4. (B) HUVECs stably overexpressing GPR4 or the control vector were pretreated with vehicle or 100 mM 29,59-dideoxyadenosine (DDA) for 1 h, followed by the treatment with indicated pH media containing vehicle or 100 mM DDA for 5 h, and the cell adhesion assay was performed as described in the ‘‘Materials and Methods’’. *, P,0.05; ***, P,0.001. (C) HUVECs stably overexpressing GPR4 or the control vector were pretreated with vehicle or SQ 22536 (1 mM) for 1 h, followed by the treatment with indicated pH media containing vehicle or 1 mM SQ 22536 for 5 h, and then the cell adhesion assay was performed. *, P,0.05; **, P,0.01. (D) HUVECs stably overexpressing GPR4 or the control vector were treated with indicated pH media containing vehicle or 500 mM 8-bromo-cAMP for 15 h (overnight), and then the cell adhesion assay was performed. (E) HUVEC/GPR4 cells were treated with indicated pH media containing vehicle or 100 ng/ml PTX for 15 h, and then the cell adhesion assay was performed. 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.g003 PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27586