ABSTRACT
Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces endothelial cell migration and proliferation in culture and is strongly angiogenic in vivo. VEGF synthesis has been shown to occur in both normal and transformed cells. The receptors for the factor have been shown to be localized mainly in endothelial cells, however, the presence of VEGF synthesis and the VEGF receptor in cells other than endothelial cells has been demonstrated. Neoangiogenesis in cartilage growth plate plays a fundamental role in endochondral ossification. We have shown that, in an avian in vitro system for chondrocyte differentiation, VEGF was produced and localized in cell clusters totally resembling in vivo cartilage. The factor was synthesized by hypertrophic chondrocytes and was released into their conditioned medium, which is highly chemotactic for endothelial cells. Antibodies against VEGF inhibited endothelial cell migration induced by chondrocyte conditioned media. Similarly, endothelial cell migration was inhibited also by antibodies directed against the VEGF receptor 2/Flk1 (VEGFR2). In avian and mammalian embryo long bones, immediately before vascular invasion, VEGF was distinctly localized in growth plate hypertrophic chondrocytes. In contrast, VEGF was not observed in quiescent and proliferating chondrocytes earlier in development. VEGF receptor 2 colocalized with the factor both in hypertrophic cartilage in vivo and hypertrophic cartilage engineered in vitro, suggesting an autocrine loop in chondrocytes at the time of their maturation to hypertrophic cells and of cartilage erosion. Regardless of cell exposure to exogenous VEGF, VEGFR-2 phosphorylation was recognized in cultured hypertrophic chondrocytes, supporting the idea of an autocrine functional activation of signal transduction in this non-endothelial cell type as a consequence of the endogenous VEGF production. In summary we propose that VEGF is actively responsible for hypertrophic cartilage neovascularization through a paracrine release by chondrocytes, with invading endothelial cells as a target. Furthermore, VEGF receptor localization and signal transduction in chondrocytes strongly support the hypothesis of a VEGF autocrine activity also in morphogenesis and differentiation of a mesoderm derived cell.
Subject(s)
Autocrine Communication , Cartilage/blood supply , Cartilage/embryology , Chondrocytes/cytology , Endothelial Growth Factors/metabolism , Lymphokines/metabolism , Neovascularization, Physiologic , Paracrine Communication , Animals , Ascorbic Acid/metabolism , Bone Development/drug effects , Bone Development/physiology , Cartilage/cytology , Cartilage/metabolism , Cell Differentiation/drug effects , Cells, Cultured , Chemotaxis/drug effects , Chick Embryo , Chondrocytes/drug effects , Chondrocytes/metabolism , Conalbumin/pharmacology , Culture Media, Conditioned/pharmacology , Endothelial Growth Factors/antagonists & inhibitors , Endothelial Growth Factors/chemistry , Endothelial Growth Factors/pharmacology , Lymphokines/antagonists & inhibitors , Lymphokines/chemistry , Lymphokines/pharmacology , Mice , Molecular Weight , Neovascularization, Physiologic/drug effects , Phosphorylation/drug effects , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Growth Factor/antagonists & inhibitors , Receptors, Growth Factor/chemistry , Receptors, Growth Factor/metabolism , Receptors, Vascular Endothelial Growth Factor , Tibia/cytology , Tibia/drug effects , Tibia/embryology , Tibia/metabolism , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50-70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.
