Recent studies have identified a specialized subset of CD31hiEMCNhi vascular endothelium that positively regulates bone formation. However, it remains unclear how CD31hiEMCNhi endothelium levels are coupled to anabolic bone formation. Mice with an osteoblast-specific deletion of Shn3, which have markedly elevated bone formation, demonstrated an increase in CD31hiEMCNhi endothelium. Transcriptomic analysis identified SLIT3 as an osteoblast-derived, SHN3-regulated proangiogenic factor. Genetic deletion of Slit3 reduced skeletal CD31hiEMCNhi endothelium, resulted in low bone mass due to impaired bone formation and partially reversed the high bone mass phenotype of Shn3−/− mice. This coupling between osteoblasts and CD31hiEMCNhi endothelium is essential for bone healing, as shown by defective fracture repair in SLIT3-mutant mice and enhanced fracture repair in SHN3-mutant mice. Finally, administration of recombinant SLIT3 both enhanced bone-fracture healing and counteracted bone loss in a mouse model of postmenopausal osteoporosis. Thus, drugs that target the SLIT3 pathway may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss.

Angiogenesis is required for bone development, growth, and repair. It is influenced by the local bone environment that involves cross-talks between endothelial cells and adjacent bone cells. However, data regarding factors that directly contribute to angiogenesis by bone cells remain poorly understood. Here, we report that EGFL6, a member of the epidermal growth factor (EGF) repeat superfamily proteins, induces angiogenesis by a paracrine mechanism in which EGFL6 is expressed in osteoblastic-like cells but promotes migration and angiogenesis of endothelial cells. Co-immunoprecipitation assays revealed that EGFL6 is secreted in culture medium as a homodimer protein.Using scratch wound healing and transwell assays, we found that conditioned medium containing EGFL6 potentiates SVEC (a simian virus 40-transformed mouse microvascular endothelial cell line) endothelial cell migration. In addition, EGFL6 promotes the endothelial cell tube-like structure formation in Matrigel assays and angiogenesis in a chick embryo chorioallantoic membrane. Furthermore, we show that EGFL6 recombinant protein induces phosphorylation of ERK in SVEC endothelial cells. Inhibition of ERK impaired EGFL6-induced ERK activation and endothelial cell migration. Together, these results demonstrate, for the first time, that osteoblastic-like cells express EGFL6 that is capable of promoting endothelial cell migration and angiogenesis via ERK activation. Thus, the EGLF6 mediates a paracrine mechanism of cross-talk between vascular endothelial cells and osteoblasts and might offer an important new target for the potential treatment of bone diseases, including osteonecrosis, osteoporosis, and fracture healing.Angiogenesis plays a pivotal role in bone formation, remodeling, and healing (1). In early osteogenesis during embryonic development, vascularization is required for the replacement of the hypertrophied cartilage core with bone marrow. During adulthood, angiogenesis is closely coupled with the process of bone remodeling (2, 3). Evidence has been presented that bone remodeling takes place in specialized vascular structures, bone remodeling compartments that contain osteoblastic-like cells and a vascular structure (4, 5). Lack of bone vascularity is associated with decreased bone formation and bone mass (1, 6). Furthermore, inhibition of angiogenesis during fracture repair in animals results in the formation of fibrous tissue and atrophic nonunions, leading to impaired bone healing (7).It has been widely speculated that bone remodeling requires an intimate connection between blood vessels and bone cells. Vascular endothelial cells, bone building osteoblasts, and bone-resorbing osteoclasts contribute multiple regulatory proteins that interplay autocrine/paracrine modes of regulation for the recruitment, proliferation, differentiation function, and survival of these vascular and bone cells (1,8). For instance, endothelial cells produce many factors that influence bone cells, including M-CSF, RANKL, 2 and chemokines (1, 9). Conversely, both osteoclas...

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