After overnight treatment with DHZ or DHZ dimer, HUVEC were harvested, stained with trypan blue 0.5% (Euroclone, Pero, MI, Italy), and counted by transmitted light microscopy using an Axioskop microscope (Carl Zeiss, Jena, Germany).
HUVEC were stained with DCFH-DA (Invitrogen Molecular Probes, Carlsbad, CA, USA) 10 [micro]M, for 30 minutes at 37[degrees]C, and then cultured in 6-well plates (Corning) (1 x [10.sup.5] cells/mL) in EBM-2 containing DHZ or DHZ dimer.
After an overnight incubation with 5 [micro]g/mL or 10 [micro]g/mL DHZ or DHZ dimer, cells were stimulated with TNF-[alpha] or medium alone for 6 hours and harvested using 0.1% Trypsin-EDTA.
To evaluate if DHZ dimer and monomer were able to modulate adhesion molecule gene expression, HUVEC (5 x [10.sup.5] cells/mL) were plated in 100 mm petri dish (Corning) and were cultured in EBM-2 containing 10 [micro]g/mL of DHZ or DHZ dimer.
To evaluate the expression of the transmembrane protein TF, HUVEC were cultured with 10 [micro]g/mL of DHZ or DHZ dimer and, after an overnight incubation, were stimulated with TNF-[alpha] (Tebu-bio) for 6 hours.
To evaluate NF-[kappa]B activation, HUVEC (1 x [10.sup.5] cells/mL) were preincubated with DHZ or DHZ dimer overnight and then treated with 10 ng/mL TNF-[alpha] (Tebu-bio) for 1 hour.
Preliminary dose-response experiments conducted to evaluate a possible toxic effect of DHZ dimer and monomer on ECs showed a reduction of cell viability after a treatment with 50 [micro]g/mL of DHZ dimer and with 100 [micro]g/mL of monomer (Figure 1).
To evaluate the antioxidant activity of DHZ and DHZ dimer on ECs we assessed the ability of these compounds to counteract ROS production induced by [H.sub.2][O.sub.2] treatment.
Cytofluorimetric analysis of adhesion molecule surface expression showed a significant reduction of TNF-[alpha]-induced ICAM-1 and VCAM-1 surface expression evaluated as MFI in response to both DHZ and DHZ dimer (Figures 3(a) and 3(b)).
Analysis of ICAM-1 and VCAM-1 concentrations in culture supernatants from TNF-[alpha]-stimulated HUVEC showed lower levels of these molecules in samples pretreated with DHZ and DHZ dimer compared to those from non-pretreated samples (Figures 5(a) and 5(b)).
In all cases, preincubation with DHZ and DHZ dimer significantly reduced such expression (Figures 6(a) and 6(b)), confirming their potential anti-inflammatory activity.
To assess the possible antithrombotic activity of DHZ and DHZ dimer, we evaluated TF expression on cell membrane.