Wang D S, Miura M, Demura H, Sato K
Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College, Shinjuku-ku, Japan.
Endocrinology. 1997 Jul;138(7):2953-62. doi: 10.1210/endo.138.7.5275.
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.
人成骨样细胞（HOB）可产生血管内皮生长因子（VEGF），其稳态水平受1,25 - 二羟维生素D3 [1,25-(OH)2D3]刺激。由于成骨细胞和内皮细胞在骨骼组织中位置相邻，我们研究了1,25-(OH)2D3和VEGF对与内皮细胞共培养的HOB的合成代谢作用。当高碱性磷酸酶（Al-P）活性的HOB与低活性的人脐静脉内皮细胞（HUVEC）共同培养时，Al-P活性增加，同时细胞数量增多。当HOB和HUVEC单独培养时，1,25-(OH)2D3不会直接刺激[3H]胸苷掺入HUVEC，但在有HOB存在时会刺激其掺入。VEGF不会直接刺激HOB的Al-P活性，但在有HUVEC存在时会刺激其活性。HOB的条件培养基可刺激HUVEC的增殖，而抗VEGF抗体可部分阻断这种增殖。相反，HUVEC的条件培养基可增加HOB的Al-P活性以及[3H]胸苷掺入量，而抗胰岛素样生长因子I抗体和内皮素-1（ET-1）受体特异性拮抗剂BQ-123可部分阻断这种增加。1,25-(OH)2D3分别刺激HOB和HUVEC释放VEGF和ET-1。此外，在与HUVEC共培养的HOB中，1,25-(OH)2D3诱导的VEGF释放增强。定量逆转录-PCR研究显示，VEGF受体（Flt-1和KDR）基因在HUVEC中表达，但在HOB中不表达，并且只有在与HOB共培养时，1,25-(OH)2D3才会增加内皮细胞中VEGF受体基因的表达水平。总之，我们证明1,25-(OH)2D3通过增强成骨细胞产生VEGF来对其发挥合成代谢作用，VEGF刺激内皮细胞上的受体，随后增加促骨生长因子如胰岛素样生长因子I和ET-1的产生。这些体外研究结果表明，VEGF/VEGF受体系统可能参与体内的骨形成和骨重塑过程。
