Crosstalk between endothelial cells and osteoblasts stimulates ALP via Notch signaling and RANKL/OPG ratio independently of Notch signaling in vitro.

BACKGROUND: Bone remodeling requires a complex interplay between osteogenesis and angiogenesis, orchestrated by yet not fully understood intricate signaling pathways in osteoblasts and endothelial cells. METHODS: In the present study, co-cultures of primary human osteoblasts and human umbilical vein endothelial cells (HUVEC) were compared with osteoblast cultures treated with dexamethasone (Dex), vascular endothelial growth factor (VEGF), their combination, or VEGF in the presence of Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). Cellular behavior was analyzed at morphological, gene expression, and protein levels to identify key regulators in the interplay between osteoblasts and endothelial cells. RESULTS: Dex and VEGF additively increased alkaline phosphatase (ALP) in osteoblast-HUVEC co-cultures, but not in osteoblast cultures. Furthermore, Dex reduced the receptor activator of nuclear factor κB ligand/osteoprotegerin (RANKL/OPG) ratio in osteoblasts. This effect was reversed in the presence of VEGF, but only in co-culture, indicating a direct action of endothelial cells, rather than VEGF itself, in stimulating RANKL and reducing OPG in osteoblasts. In addition, Notch signaling, specifically NOTCH1 and DLL4, was induced in response to VEGF solely in co-cultures. The presence of Notch inhibitor DAPT suppressed VEGF-induced stimulation of ALP but not RANKL/OPG ratio. CONCLUSIONS: Our findings provide novel evidence for the significant role of endothelial cells in bone remodeling, specifically in regulating ALP expression and activity of osteoblasts via the Notch signaling pathway and RANKL/OPG ratio independent of Notch. This study underscores the applicability and significance of multicellular tissue models for studying bone turnover processes in vitro, thereby reducing the reliance on animal testing.