LPA receptors LPA1 and LPA3 regulate CD36 MVEC expression. (A) Lysates prepared from confluent cultures of human umbilical vein endothelial cells (HUVECs), HMVECs and 2 strains of murine B16 melanoma cells were analyzed by Western blot using an antibody that recognizes LPA1 (top band) and LPA3 (bottom band). Blots were stripped and reprobed with anti–β-actin as a loading control. (B) Matrigel containing FGF-2 was injected subcutaneously into mice, and after 10 days the plugs were removed, sectioned, and analyzed by indirect immunofluorescence microscopy with a Leica DM-RXE microscope equipped with a 10×/0.30 NA objective using antibodies to LPA3 (green) or VE-cadherin (red). A representative merged image is shown in the third panel. The far right image shows a control (CTL) with secondary antibody alone. Scale bar = 100 μm. (C) HMVECs were pretreated with the LPA1,3 antagonist Ki14625 (Ki; 2μM) or vehicle control followed by LPA (5μM) for 24 hours. Quantitative real-time PCR of CD36 mRNA was then done as in Figure 1D. Data are expressed relative to a control transcript, PPIA. The mean value for untreated samples was set to 1, and the bar graph shows mean ± SEM of 3 independent experiments.
