Interaction of clodronate with fibroblast growth factor 2 reduces FGF2-activity in endothelial cells

Stabilization of fibroblast growth factors from heat denaturation and proteolytic digestion by bound heparin and heparan sulphate proteoglycans is known for more than 20 years. Furthermore, ATP-binding to FGF2 also leads to stabilization of this growth factor as discovered recently. The physiological importance of this protection is not yet clear but has become the focal point of interest. In this study we used the method of stabilizing FGF2 from proteolytic degradation to identify some bisphosphonates, namely clodronate and etidronate, which interact with FGF2. These two bisphoshonates protect FGF2 from tryptic digestion in vitro. The circular dichroism spectrum of FGF2 incubated with clodronate was significantly shifted compared to the spectrum of non-treated FGF2 indicating a conformational change of the protein after clodronate-binding. Additionally, clodronate and etidronate at low μM concentrations induce a concentration-dependent reduction of FGF2-induced cell proliferation of human umbilical vein endothelial cells. In contrast, proliferation of these cells after addition of clodronate and etidronate without FGF2 was not influenced by these bisphosphonates. Furthermore, the intracellular signaling via ERK1/2 and AKT was inhibited by clodronate and the tube formation, indicating the beginning process of angiogenesis, was reduced. Our results show for the first time that bisphosphonates I) interact with FGF2, II) reduce FGF2-activity and III) decrease the angiogenic potential of this growth factor.