title: Bone morphogenetic proteins stimulate angiogenesis through osteoblast-derived vascular endothelial growth factor A.
abstract: During bone formation and fracture healing there is a cross-talk between endothelial cells and osteoblasts. We previously showed that vascular endothelial growth factor A (VEGF-A) might be an important factor in this cross-talk, as osteoblast-like cells produce this angiogenic factor in a differentiation-dependent manner. Moreover, exogenously added VEGF-A enhances osteoblast differentiation. In the present study we investigated, given the coupling between angiogenesis and bone formation, whether bone morphogenetic proteins (BMPs) stimulate osteoblastogenesis and angiogenesis through the production of VEGF-A. For this we used the murine preosteoblast-like cell line KS483, which forms mineralized nodules in vitro, and an angiogenesis assay comprising 17-d-old fetal mouse bone explants that have the ability to form tube-like structures in vitro. Treatment of KS483 cells with BMP-2, -4, and -6 enhanced nodule formation, osteocalcin mRNA expression, and subsequent mineralization after 18 d of culture. This was accompanied by a dose-dependent increase in VEGF-A protein levels throughout the culture period. BMP-induced osteoblast differentiation, however, was independent of VEGF-A, as blocking VEGF-A activity by a VEGF-A antibody or a VEGF receptor 2 tyrosine kinase inhibitor did not affect BMP-induced mineralization. To investigate whether BMPs stimulate angiogenesis through VEGF-A, BMPs were assayed for their angiogenic activity. Treatment of bone explants with BMPs enhanced angiogenesis. This was inhibited by soluble BMP receptor 1A or noggin. In the presence of a VEGF-A antibody, both unstimulated and BMP-stimulated angiogenesis were arrested. Conditioned media of KS483 cells treated with BMPs also induced a strong angiogenic response, which was blocked by antimouse VEGF-A but not by noggin. These effects were specific for BMPs, as TGF beta inhibited osteoblast differentiation and angiogenesis while stimulating VEGF-A production. These findings indicate that BMPs stimulate angiogenesis through the production of VEGF-A by osteoblasts. In conclusion, VEGF-A produced by osteoblasts in response to BMPs is not involved in osteoblast differentiation, but couples angiogenesis to bone formation.
authors.affiliation: Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
authors.forename: Martine M L
authors.initials: MM
authors.lastname: Deckers
authors.forename: Rutger L
authors.initials: RL
authors.lastname: van Bezooijen
authors.forename: Geertje
authors.initials: G
authors.lastname: van der Horst
authors.forename: Jakomijn
authors.initials: J
authors.lastname: Hoogendam
authors.forename: Chris
authors.initials: C
authors.lastname: van Der Bent
authors.forename: Socrates E
authors.initials: SE
authors.lastname: Papapoulos
authors.forename: Clemens W G M
authors.initials: CW
authors.lastname: Löwik
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D017077:Culture Media, Conditioned; D003609:Dactinomycin / Q000494:pharmacology; D016228:Endothelial Growth Factors / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013268:Stimulation, Chemical; D014158:Transcription, Genetic / Q000187:drug effects; D042461:Vascular Endothelial Growth Factor A
chemical_list: D019485:Bone Morphogenetic Proteins; D017077:Culture Media, Conditioned; D016228:Endothelial Growth Factors; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D003609:Dactinomycin; D002118:Calcium