Osteoprotegerin and Adhesion Molecules - Adhesion Molecules - page 272

that topical administration of OPG to rat mesenteric post-capillary venules
increased leukocyte rolling. h ese studies strongly suggest that elevated levels
of OPG contribute to inl ammation. h e investigators also reported that TNF-α
induced production of OPG from endothelial cells, suggesting a link between
mechanisms promoting inl ammation (Zauli et al. 2007).
At approximately the same time as Zauli et al. (2007) reported their i ndings we
reported a similar study of the inl uence of OPG on leukocyte adhesion (Mangan
et al. 2007). In line with Zauli and colleagues, we reported that incubating
HUVECs with OPG alone (0.5-10 ng/ml) had no inl uence on expression of a
range of adhesion molecules. In contrast, OPG stimulated a dose-dependent
increase in intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion
molecule 1 (VCAM-1) and E-Selectin (CD62E) messenger ribonucleic acid
(mRNA) and surface protein expression in HUVECs activated with TNF-α
compared to control cells (Fig. 1) (Mangan et al. 2007). h e maximal ef ect was
seen at an OPG concentration of approximately 10 ng/ml. In keeping with this
i nding, we demonstrated that in the presence of TNF-α (50 U/ml), OPG (0.5-10
ng/ml for 12 hr) stimulated a dose-dependent increase in binding of a monocyte
cell line (THP-1) to HUVECs (Fig. 2) (Mangan et al. 2007). h e latter i ndings are
not in agreement to those reported by Zauli and colleagues, who reported no co-
stimulatory ef ect of OPG and TNF-α on adhesion of PMNs to HUVECs (Zauli
et al. 2007). h ese dif erences may relate to cell types used, incubation times or
doses of OPG, all of which dif ered between experiments (Mangan et al. 2007,
Zauli et al. 2007). In contrast to the TNF-α−related ef ects, we found that OPG did
not augment adhesion molecule expression in Interleukin-1 beta (IL-1b) activated
endothelial cells. Our overall i ndings suggested a TNF-α- and OPG-specii c
cooperation in promoting leukocyte adhesion.
In order to investigate the mechanisms that might be responsible for the
upregulation of adhesion molecules in TNF-α-activated HUVECs, we studied
gene expression in resting HUVECs using expression arrays and showed that
angiopoietin-2 (Ang-2) expression was signii cantly altered following OPG
incubation (Fig. 3) . Using quantitative gene expression analysis (MassArray), we
found that incubating HUVECs with 10 ng/ml concentrations of OPG for 4 hr
induced a two-fold increase in Ang-2 gene expression (162.3 ± 26.9 fM compared
to 88.2 ± 8.9 fM in control cells). We found increased Ang-2 protein within Weibel-
Palade bodies in OPG-treated resting HUVECs (Fig. 4) .
Fiedler et al . (2006) have shown that Ang-2 facilitates leukocyte adhesion by
potentiating the TNF-α−mediated expression of the adhesion proteins ICAM-1
and VCAM-1. Ang-2-dei cient mice have impaired inl ammatory responses to
some stimuli. Studies in cell culture by Fiedler et al . (2006) showed that Ang-2
sensitized cells to the ef ect of TNF-α. In particular, these investigators showed
that Ang-2 augmented the ability of TNF-α to upregulate adhesion molecules.