Figure 7.

ATX regulates HUVEC permeability via a LPA₁-independent mechanism. (a) Relative mRNA levels of LPA receptors in HUVECs. ΔΔCT method was used to calculate relative gene expression of LPA receptors with β2MG being the internal control. Data are expressed as mean ± SEM. (b) The addition of ATX and LPC to HUVECs enhances permeability in an ATX concentration-dependent manner. (n = 3 cell preparations/group). (c) Pre-treatment with 5 nM PAT-048, but not 10 nM AM152, suppresses endothelial cell permeability induced by ATX and LPC (n = 3 cell preparations/group). Data from (b,c) are expressed as mean ± SEM. (d) Actin polymerization was visualized by immunocytochemical staining for phalloidin in HUVECs that had been incubated in media containing 0.5% FBS for 2 hours and then stimulated for 30 min with 10 nM ATX added to 10 μM LPC or 10 μM LPC alone. HUVECs were pre-treated with 5 nM PAT-048 or 10 nM AM152 for 30 min. Arrows indicate intercellular gaps. Bars, 100 μm. All images were captured using identical exposure settings. (e) The number of intercellular gaps induced by the addition of ATX with LPC was inhibited by 5 nM PAT-048, but not 10 nM AM152. Four different fields were counted. Data are expressed as mean ± SEM.