Figure 10.

siRNA knockdown of HO-1 blocked the inhibitory action of EGCG on TNF-α-stimulated VCAM-1 expression and monocyte adhesion in endothelial cells. A. HAEC were transiently transfected with scrambled siRNA (control) or siRNA targeting HO-1 as described in Methods. 48 h after transfection, cells were serum-starved for 1 h and then treated with vehicle or TNF-α (10 ng/ml) for 5 h in the presence or absence of EGCG (2.5 μM). Cell lysates were then subjected to immunoblotting using antibodies against VCAM-1, HO-1, HO-2, and β-actin as indicated. Immunoblots shown are representative of experiments that were repeated independently 4 times. B. Results for VCAM-1 expression from 4 independent experiments represented in panel A were quantified by scanning densitometry and normalized to β-actin expression (mean ± SEM). In cells transfected with control scrambled siRNA, EGCG treatment diminished TNF-α stimulated VCAM-1 expression (p < 0.001). In cells transfected with HO-1 siRNA, EGCG treatment failed to modulate TNF-α induced VCAM-1 expression with, (p > 0.05) (by ANOVA and Bonferroni’s post-test). C. HAEC were transfected with scrambled siRNA (control) or siRNA specifically targeting HO-1, p38-MAPK, or nrf-2. 48 h after transfection, cells were serum-starved for 1 h and then treated with vehicle or TNF-α (10 ng/ml) for 5 h in the presence or absence of EGCG (2.5 μM). Labeled U937 monocytes (6 × 10⁵) were incubated for 30 min at 37 °C with confluent HAEC pre-treated with EGCG and/or TNF-α. Co-cultured cells were washed 3 times with PBS and fixed in 2% folmaldehyde. Images of monocytes adhering to HAECs were visualized using an epifluorescent microscope (upper panel). Phase contrast view of cells is shown in lower panel.