BRONJ: The effects of zoledronic acid on the VEGF receptors and implications for osteoblast growth and maturation

Background: The pathogenesis of bisphosphonate related osteonecrosis of the jaw (BRONJ) is not well understood, although its clinical presentation and response to treatment reflect a failure to heal. Vascular endothelial growth factor (VEGF) is involved in both angiogenesis and osteogenesis and plays a role in tissue formation, modelling and remodelling in response to injury. VEGF is upregulated in zoledronic acid (ZA) treated cells and therefore any inhibition of this pathway in BRONJ may be via the VEGF receptors. Protein prenylation is essential for VEGF receptor intracellular transportation and anchorage to the cell membrane. The mevalonate pathway (MVP) is an intracellular target for the nitrogen bisphosphonates and disruption prevents protein prenylation. Aim: To determine the role of the VEGF receptors (R1 and R2) in human alveolar osteoblast (HOB) growth and maturation and to investigate receptor protein expression in the presence and absence of ZA and a downstream MVP metabolite, geranylgeraniol (GGOH). Hypothesis: VEGF receptor inhibition will affect mineralised nodule formation by HOBs and the mechanism of this effect is via inhibition of the MVP. Materials and methods: An alizarin red mineralisation assay was used to quantitatively analyse calcium deposition by HOBs after 21 and 28 days of culture in the presence of a VEGFR1, VEGFR2 or a dual VEGFR1/VEGFR2 inhibitor. Alizarin red and alkaline phosphatase staining were used to identify the presence of nodules under these conditions at 21 days of culture. HOBs were cultured and treated with various combinations of VEGF, ZA and GGOH for 48 h. Immunofluorescence and confocal microscopy were used to analyse VEGFR1 protein expression under these conditions. Results: VEGFR1 and dual VEGFR1/VEGFR2, but not VEGFR2 inhibition lead to a dose dependent decrease in mineralisation by human alveolar osteoblasts. The present experiment did not detect a difference in VEGFR1 protein expression by HOBs treated with combinations of ZA, VEGF and the mevalonate pathway constituent GGOH. Conclusion: The VEGF/VEGFR1 pathway is important, but not essential to osteoblast growth and maturation in vitro. The mechanism of VEGFR1 inhibition by the potent nitrogen bisphosphonate, ZA, remains to be elucidated