Expression of Notch receptors in endothelial cells and Notch function in Dll4-transduced cells. (A) mRNAs for Notch receptors 1 to 4 detected by RT-PCR in HUVECs transduced with vector. (B) Quantitative RT-PCR analysis of HEY1, HEY2, ephrinB2, and EphB2 expression in control HUVECs transduced with vector only or Dll4-overexpressing HUVECs. The results reflect relative mRNA levels (normalized to GAPDH) and are expressed as the mean (± SEM) fold mRNA change in Dll4 versus control HUVECs from 2 to 11 separate determinations. ^*P < .05. (C) Effects of immobilized soluble recombinant human Dll4 (rhDLL4) on the proliferation of HUVECs. The endothelial cells were cultured for 72 hours in the presence of VEGF-A (25 ng/mL) on plates coated with rhDLL4 (1 μg/mL) or diluent (0.1% BSA); proliferation was measured by ³H thymidine deoxyribose uptake during the last 18 hours of culture. The results reflect the means (± SDs) from triplicate cultures. Representative experiment of 4 performed. ^*P < .05. (D) Surface levels of VEGFR2 expression in HUVECs cultured for 48 hours onto rhDLL4-coated plates (1 μg/mL) (rhDLL4) or diluent-coated plates (gelatin) measured by flow cytometry. Control reflects background surface fluorescence staining with appropriate controls. Representative of 2 performed. (E) Effects of the γ-secretase inhibitor L-685458 (0.1-7.5 μM) on the proliferation of HUVECs cultured with rhDLL4-coated plates (1 μg/mL); proliferation was measured by [³H] thymidine deoxyribose uptake during the last 18 hours of culture. The results reflect the means (± SDs) from triplicate cultures. Representative experiment of 4 performed. ^*P < .01 (rhDLL4 with 4 μM L-685458 inhibitor versus VEGF alone). (F) Quantitative RT-PCR analysis of GAPDH and VEGFR2 expression in vector- and Dll4-transduced HUVECs after 72-hour culture with L-685458 (2 μM) or diluent only (0.02% DMSO). The results reflect relative mRNA levels (normalized to GAPDH) and are expressed as the mean (± SEM) fold mRNA change in Dll4-transduced versus control HUVECs from 4 separate experiments, in which levels of VEGFR2 expression in the absence of inhibitor were reduced by at least 5-fold in Dll4-transduced compared with control HUVECs (^*P < .05).