FIGURE 1.

Egfl7 is repressed in endothelial cells under inflammatory conditions in vivo. A, left panel, in situ hybridization detection of Egfl7 transcripts in endothelial cell nuclei of adult mouse lungs (blue staining, arrows). Right panel and inset, CD31 immunostaining (brown, arrows) and hematoxylin counterstaining of a parallel section of the same area. Bar, 25 μm. B, expression levels of CD31 and Egfl7 transcripts in CD31⁻ cells (white bars) and CD31⁺ cells (black bars) isolated from mouse lungs using immunoaffinity and measured by duplex RT-qPCR using a mouse CD31-FAM or a mouse Egfl7-FAM TaqMan probe mixed with a mouse β-actin-VIC probe (see “Experimental Procedures”). The results are plotted as quantities relative to CD31⁻ controls values set to 1. RQ, relative quantities. C, LPS (5 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils, and animals were sacrificed at the onset of treatment (0 h) or after 10 or 24 h; the lungs were dissected and processed for total RNA isolation. Expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 were measured by duplex RT-qPCR using the indicated FAM-labeled TaqMan probe for the mouse transcript of interest and a mouse β-actin-VIC-labeled TaqMan probe and expressed as 2^−ΔΔCT quantities relative to t = 0 h values set to 1. *, p < 0.05; **, p < 0.01; ***, p < 0,001. The results are representative of three experiments performed in triplicate. RQ, relative quantities. D, left panel, HUVEC were treated with increasing doses of LPS for 4 h and Egfl7 transcript levels assessed by duplex RT-qPCR. Right panel, expression levels of Egfl7 transcripts in primary HUVEC cells treated with 0.1 μg/ml of LPS for the indicated length of time and assessed by duplex RT-qPCR. E, TNFα (0.25 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils and lungs processed as in C for the analysis of expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 expressed as relative to t = 0 values set to 1. RQ, relative quantities. **, p < 0.01; ***, p < 0,001.