The effect of nitrogen-containing bisphosphonates, alendronate and zoledronate, on PDLSCs-induced angiogenesis

Alendronate (ALN) and zoledronate (ZOL) are two potent amino-bisphosphonate (N-BPs) widely used for cancer indications and osteoporosis [1]. At the cellular level, N-BPs mainly exert their effects on osteoclasts, however, their impact on osteoblasts, endothelial and mesenchymal cells has also been described. At the molecular level, N-BPs inhibit the action of farnesyl pyrophosphate synthase in the mevalonate pathway, thereby blocking the prenylation of small GTPases that are major regulators of intracellular trafficking [2]. When N-BPs bind to bone, they localize preferentially at sites of resorption, where osteoblasts and endothelial cells are exposed to a much lower concentration in vivo than the osteoclasts. Although the association of N-BPs and osteonecrosis of the jaw (MRONJ) has been widely described, pathophysiology of the MRONJ has not been elucidated. We and others reported dose-dependent effects of ALN and ZOL on periodontal ligament stem cells (PDLSCs), which play a key role in periodontal tissue remodeling [3,4]. Recently, more attention has been directed to the role of N-BPs-induced vascular alteration in the initiation and progression of the disease. With this aim, we evaluated the release of VEGF and MCP-1, two important angiogenic growth factor and chemokine, respectively, in the supernatant of PDLSCs exposed to increasing doses of ALN (2 μM, 5 μM, 10 μM,) and ZOL (1 μM, 1,5 μM, 2 μM) for 24 h, 48 h, and 120 h. Then, we investigated if those modulations have any role in impairing PDLSCs-induced angiogenesis, via tube formation assay. By microarray analysis, we recorded increased VEGF and MCP-1 levels in the medium of PDLSCs after exposure to 1 M ZOL compared to unexposed cells. Conversely, a reduction of VEGF and MCP-1 release was reported after exposure to 1,5 M and to a lesser extent to 2 M ZOL at all times considered, compared to cells exposed to 1 M ZOL. Similarly, an increase in both VEGF and MCP-1 was reported after exposure to 2 M ALN at all time points tested. Also in this case, exposure to the higher ALN concentrations (5 and 10 M) accounted for a reduction in VEGF and MCP-1 production compared to cells exposed to 2 M ALN. Tube-formation capacity of HUVECs were then tested in the presence of supernatant from PDLSCs +/- N-BPs. Quantitative analysis was performed by ImageJ software, and we found that both ALN and ZOL were able to modulate the number of meshes, nodes, and total segment length in a dose-dependent manner, compared with conditioned medium without drug. Although additional studies are needed, our study suggest new insights on modulatory action of N-BPs, ALN and ZOL, in PDLSCs-induced angiogenesis.