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Anabolic Effects of 1,25-Dihydroxyvitamin D₃ on Osteoblasts Are Enhanced by Vascular Endothelial Growth Factor Produced by Osteoblasts and by Growth Factors Produced by Endothelial Cells¹

Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women’s Medical College (D.S.W., H.D., K.S.), Shinjuku-ku; and Mitsubishi Kagaku BCL (M.M.), Itabashi-ku, Tokyo, Japan

Address all correspondence and requests for reprints to: Kanji Sato, M.D., Institute of Clinical Endocrinology, Tokyo Women’s Medical College, Kawada-cho 8–1, Shinjuku-ku, Tokyo 162, Japan.

Human osteoblast-like cells (HOB) produce vascular endothelial growth factor (VEGF), the steady state level of which is stimulated by 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃]. As osteoblasts and endothelial cells are proximally located in skeletal tissue, we investigated the anabolic effects of 1,25-(OH)₂D₃ and VEGF on HOB cocultured with endothelial cells.

When HOB with high alkaline phosphatase (Al-P) activity and human umbilical vein endothelial cells (HUVEC) with little activity were cultured together, Al-P activity increased, accompanied by an increase in cell number. When HOB and HUVEC were cultured separately, 1,25-(OH)₂D₃ did not directly stimulate [³H]thymidine incorporation into HUVEC, but stimulated it in the presence of HOB. VEGF did not directly stimulate the Al-P activity of HOB but stimulated it in the presence of HUVEC. The conditioned medium of HOB stimulated the proliferation of HUVEC, and this was partially blocked by anti-VEGF antibody. Conversely, the conditioned medium of HUVEC increased Al-P activity and [³H]thymidine incorporation into HOB, and this was partially blocked by antiinsulin-like growth factor I antibody and BQ-123, a specific antagonist of the endothelin-1 (ET-1) receptor. 1,25-(OH)₂D₃ stimulated the release of VEGF and ET-1 from HOB and HUVEC, respectively. Furthermore, the 1,25-(OH)₂D₃-induced release of VEGF was enhanced in HOB cocultured with HUVEC. A quantitative reverse transcription-PCR study revealed that genes for VEGF receptors (Flt-1 and KDR) were expressed in HUVEC, but not in HOB, and that 1,25-(OH)₂D₃ increased the levels of expression of VEGF receptor genes in endothelial cells only when cocultured with HOB.

In summary, we demonstrated that 1,25-(OH)₂D₃ exerts an anabolic effect on osteoblasts by enhancing their production of VEGF, which stimulates its receptors on endothelial cells, followed by increased production of osteotropic growth factors, such as insulin-like growth factor I and ET-1. These in vitro findings suggest that the VEGF/VEGF receptor system may be involved in both bone formation and bone remodeling in vivo.

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