H₂O₂ induced senescent ECs are non inflammatory. HUVECs were stimulated with 100μM H₂O₂ for 2 hours and then placed in normal HUVEC medium for 24 hours. They were then replated into Labtek slides and 24 hours later stimulated with 5 ng/mL TNFα for 5 hours then stained for E-selectin (A) or VCAM1 (B) (right panels); Bar = 220 μm. Phase contrast photographs of the area for adhesion molecule expression are given in the left panels. (C) Senescent and nonsenescent cells were analyzed for cell surface expression of E-selectin and VCAM1. The mean pixel intensity per cell was measured using ImageJ for senescent cells (■) and for neighboring nonsenescent cells (□). The E-selectin data are a representative of the mean ± SEM of 33 senescent cells and nonsenescent cells from 2 HUVEC lines. ***P < .001 compared with nonsenescent cells. The VCAM1 data are a representative of the mean ± SEM of 17 senescent cells and the corresponding area of nonsenescent cells from 2 HUVEC lines. ***P < .001 compared with nonsenescent cells. (D) Total RNA was extracted from the aortas of male ApoE^−/− mice that were on a Western diet for 1 (□) and 5 months (■). The RNA was subjected to reverse transcriptase followed by Q-RT-PCR, as described in Methods. mRNA expression of Senex was assessed and standardized to Pbgd, Hrpt, and Tie2. Quantification of Senex levels was standardized to the 3 housekeeping genes.