Osteoprotegerin (OPG) a soluble tumor necrosis factor receptor family molecule protects endothelial cells from apoptosis in vitro and promotes neovascularization in vivo. In this study, we assessed the role of OPG and its ligands, receptor activator of nuclear factor-kappaB ligand (RANKL) and tumor necrosis factor-related apoptosis inducing ligand (TRAIL), in microvessel formation using the rat aortic ring model of angiogenesis. OPG was found to promote a twofold increase in angiogenic sprouting in the aortic ring model, and this effect was inhibited by pre-incubation with a fivefold molar excess of either RANKL or TRAIL. While TRAIL had no effect upon angiogenesis on its own, RANKL was found to potently inhibit basal and vascular endothelial growth factor-induced angiogenesis. OPG increased the rate of endothelial cell proliferation in sprouting microvessels; in contrast, RANKL inhibited proliferation. RANKL was found to induce endothelial apoptosis at days 6, 7, and 10 in the aortic ring model and after incubation with human umbilical vein endothelial cells (HUVECs). Signaling studies showed that OPG induced ERK1/2 and Akt phosphorylation in HUVECs while RANKL had no effect. Our results indicate that OPG is a positive regulator of microvessel formation, while RANKL is an angiogenic inhibitor due to effects on regulation of endothelial cell proliferation, apoptosis, and signaling.

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Localized, controlled delivery of pro-angiogenic agents is an important component of therapies for chronic wound treatment and regenerative medicine. Osteoprotegerin (OPG), a tumor necrosis factor receptor (TNFR) superfamily member has recently been shown to be pro-angiogenic in vitro, and we hypothesized that controlled delivery of OPG could induce angiogenesis in vivo. OPG contains a highly basic heparin-binding domain, suggesting that affinity interactions could be used to control the rate of its ion-exchange-driven release from drug-delivery devices. Here, we describe the use of a hydrogel consisting of thiol-modified heparin which can be readily and controllably cross-linked using a bi-functional PEG-diacrylate. These hydrogels were found to retain between 750 and 900 ng of immunoreactive OPG for up to 500 h in in vitro release studies. OPG containing hydrogels were evaluated in a subcutaneous mouse implant model, and exhibited little degradation and retained OPG as indicated by immunohistochemistry at 2 weeks post-implantation. Immunohistochemical analysis of implanted gels indicated that OPG induced nearly a 2-fold increase in vascular density in the surrounding foreign body capsule. These results suggest that the controlled delivery of OPG can stimulate angiogenesis in vivo and may be of use for wound healing therapies as well as other inflammatory and bone disorders in which OPG plays a role.