Figure 4

(a) Expression of Notch pathway proteins in proliferating and confluent EC. Western blot analysis of proliferating (Prol) or confluent (Conf) EC proteins from four donors (two are shown) revealed a 1.5–2.0-fold decrease in transmembrane Notch-1 and virtually absent transmembrane Notch-2 expression in confluent cells compared to ample expression in proliferating EC. There was little to no activated Notch-1 or Notch-2 in either cell population. Activated Notch-4 levels were slightly decreased (range: 1.2–1.5-fold) in confluent EC. The Notch ligand Dll-1 was modestly expressed and unchanged between proliferating and confluent cells, while Jagged-1 expression was significantly decreased upon confluency. Hey-1 expression was similar or decreased in confluent cells, while p21 expression was significantly decreased. In contrast, Hes-1 expression was consistently upregulated 1.6–2.0-fold in confluent cells. (b) Luciferase reporter activity in proliferating and confluent EC. There was a two-fold decrease (P<0.05) in Hey-1 promoter driven luciferase activity in confluent EC compared to proliferating cells. In contrast, there was a four-fold increase (P<0.05) in Hes-1 driven activity in confluent vs proliferating EC. (c) γ-secretase inhibition blocked Notch expression and/or activation in proliferating and confluent EC; however, Hes-1 expression remained elevated. Hes-1 expression was similar in confluent EC treated with the γ-secretase inhibitor or treated with DMSO as a control. Hes-1 levels were significantly induced in proliferating cells treated with the inhibitor, although the responsible mechanism has not yet been determined. (d) Induction of Hes-1 in quiescent EC is transcriptionally regulated. Quantitative, real-time RT-PCR was used to measure Hes-1 mRNA expression in proliferating and confluent EC in four different EC donors (two are shown). Hes-1 mRNA levels were normalized to GAPDH expression and the relative quantity of Hes-1 mRNA shown. Although overall levels of Hes-1 mRNA varied between individual EC donors, there was a consistent increase in Hes-1 mRNA in confluent EC compared to proliferating EC (P<0.01).