Fig. 5
Deletion of Notch1 results in regression of cell alignment, compromised cell–cell junctions, and enhanced proliferation. a Schematic to illustrate time line for in vivo model of inducible deletion of endothelial Notch1 (Notch1 ECKO). Cre+ Notch1 loxP/loxP mice after 8 weeks of age received three intraperitoneal injections of tamoxifen as did age-matched control mice at specified time points. b Notch1 transcript levels from descending aortae of adult WT mice and Notch1 ECKO mice 2 weeks post tamoxifen measured by qPCR analysis (n = 12 WT and n = 5 Notch1 ECKO). T-test ****P < 0.0001. c En face confocal imaging of the endothelium of the descending aorta for Notch1 ECKO, 1 week post deletion, and control mice (Cre− Notch1 loxP/loxP); all mice were injected with tamoxifen. Ve-Cadherin revealed instances of endothelial cells oriented perpendicular to blood flow direction (highlighted by dashed ovals). Quantification of cell orientation, measured by elongation factor (cell length along flow direction divided by cell width) for Notch1 ECKO vs. control (Cre− Notch1 loxP/loxP). All mice were injected with tamoxifen and evaluated 1 week later (100 cells were measured from 5 mice for each genotype, T-test ****P < 0.0001). d En face imaging of descending aortas 2 weeks post tamoxifen revealed fibrinogen deposited between endothelial cells (open arrow heads) in Notch1 ECKO animals, but not in age-matched tamoxifen-injected controls. Determination of fibrinogen was obtained by mean fluorescence intensity in areas of 500 µm2 from five mice of each genotype. Graph bars represent mean ± SEM. T-test *P < 0.05. e Proliferating endothelial cells, as evidenced by the mitotic figures (solid arrow heads), in Notch1 ECKO aortas but not in control (Cre− Notch loxP/loxP). f Experimental design to quantify endothelial proliferation by EdU incorporation in the descending aortae of control and Notch1 ECKO animals at 2 weeks post tamoxifen. g Representative en face images and quantification of EdU-positive endothelial cells per unit area of aorta (EdU+ endothelial cells per mm2). Notch1 ECKO and control (Cre+ tdTomato reporter). EdU incorporation is visualized in green. Graph bars represent mean of ~2 cm2 segments of the aorta evaluated from six mice per genotype ± SEM. T-test ***P < 0.001. Scale bars = 20 µm
