INTRODUCTION

Studies have suggested that OPG promotes leukocyte adhesion, although the mechanisms involved are currently controversial (Mangan et al. 2007, Zauli et al. 2007). Zauli et al. (2007) found that incubating cultured human umbilical vein endothelial cells (HUVECs) and human microvascular endothelial cells (HMVECs) with recombinant OPG for 16 hr promoted the adhesion of primary polymorphonuclear neutrophils (PMNs; drawn from healthy volunteers) and the cell line HL-60 to endothelial cells. Adhesion of leukocytes was maximally stimulated at concentrations between 0.1 and 0.5 ng/mL, which are similar to levels reported in the seraof patients aff ected by a number of diseases associated with infl ammation, such as rheumatoid arthritis (Geusens et al. 2006), symptomatic atherosclerosis (Golledge et al. 2004, Van Campenhout and Golledge 2009) and abdominal aortic aneurysm (Moran et al. 2005).Zauli and colleagues found that incubating endothelial cells with OPG alone had no eff ect on adhesion molecule expression. Th ese investigators also reported that in the presence of the proinfl ammatory cytokine TNF-α, OPG (0.5 ng/ml) did not promote additional leukocyte adhesion (Zauli et al. 2007). Further experiments by Zauli et al. suggested that incubation periods as short as 5 min were enough to stimulate increased adhesion of leukocytes to endothelial cells. Furthermore, the eff ects of OPG could be stimulated by incubation with either the PMNs or the endothelial cells. Further experiments provided additional data suggesting that OPG bound to PMNs via its ligand-(RANKL and TRAIL) binding domain, while endothelial cells bound to OPG via its heparin-binding domain (Zauli et al. 2007). Th is would suggest that circulating OPG acts as a bridge between the PMNs and endothelial cells, facilitating leukocyte rolling and fi rm adhesion. Th e investigators also reported

that topical administration of OPG to rat mesenteric post-capillary venules increased leukocyte rolling. Th ese studies strongly suggest that elevated levels of OPG contribute to infl ammation. Th e investigators also reported that TNF-α induced production of OPG from endothelial cells, suggesting a link between mechanisms promoting infl ammation (Zauli et al. 2007). At approximately the same time as Zauli et al. (2007) reported their fi ndings we reported a similar study of the infl 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 infl 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). Th e maximal eff ect was seen at an OPG concentration of approximately 10 ng/ml. In keeping with this fi 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). Th e latter fi ndings are not in agreement to those reported by Zauli and colleagues, who reported no costimulatory eff ect of OPG and TNF-α on adhesion of PMNs to HUVECs (Zauli et al. 2007). Th ese diff erences may relate to cell types used, incubation times or doses of OPG, all of which diff ered between experiments (Mangan et al. 2007, Zauli et al. 2007). In contrast to the TNF-α−related eff ects, we found that OPG did not augment adhesion molecule expression in Interleukin-1 beta (IL-1b) activated endothelial cells. Our overall fi ndings suggested a TNF-α-and OPG-specifi 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 signifi 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 WeibelPalade 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-defi cient mice have impaired infl ammatory responses to some stimuli. Studies in cell culture by Fiedler et al. (2006) showed that Ang-2 sensitized cells to the eff ect of TNF-α. In particular, these investigators showed that Ang-2 augmented the ability of TNF-α to upregulate adhesion molecules.