ARTICLES https://doi.org/10.1038/s41591-018-0020-z Targeting skeletal endothelium to ameliorate bone loss Ren Xu 1, Alisha Yallowitz1, An Qin2, Zhuhao Wu3, Dong Yeon Shin1, Jung-Min Kim4, Shawon Debnath1, Gang Ji5,6, Mathias P. Bostrom5,7, Xu Yang5, Chao Zhang8, Han Dong9,10, Pouneh Kermani11, Sarfaraz Lalani1, Na Li1, Yifang Liu1, Michael G. Poulos11, Amanda Wach12, Yi Zhang1, Kazuki Inoue13,14, Annarita Di Lorenzo1, Baohong Zhao13,14, Jason M. Butler11, Jae-Hyuck Shim 4, Laurie H. Glimcher9,10* and Matthew B. Greenblatt1* Recent studies have identified a specialized subset of CD31hiendomucinhi (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 because of impaired bone formation and partially reversed the high bone mass phenotype of Shn3−/− mice. This coupling between osteo- blasts 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 path- way may represent a new approach for vascular-targeted osteoanabolic therapy to treat bone loss. t is increasingly appreciated that the ancillary cell types present to form mature bone. Given the substantial osteoblast-intrinsic aug- in bone tissue actively participate in osteogenesis. For example, mentation of bone formation in SHN3-deficient mice, we believed Ia subset of CD31hi, endomucinhi (EMCNhi) vascular endothe- that this mouse strain represented an ideal opportunity to study lium has been recently identified as residing in the bone mar- how osteoblasts induce changes in their supporting cell lineages to row (BM) near the growth plate. CD31hiEMCNhi endothelium is facilitate bone formation. believed to actively direct bone formation, because alterations In this article, we show that CD31hiEMCNhi skeletal vasculature in CD31hiEMCNhi endothelium impact bone architecture, bone is present at greater levels in mice with an inducible deletion of Shn3 formation and numbers of osteoprogenitors present in the mar- in osteoblasts and demonstrate that CD31hiEMCNhi endothelium is row1,2. CD31hiEMCNhi endothelium levels can be altered by regulated by osteoblasts. We further identify the secreted ligand platelet-derived growth factor type BB (PDGF-BB), providing a SLIT3 as a novel SHN3-controlled, osteoblast-derived regulator of potential link between bone resorption activity by osteoclasts and CD31hiEMCNhi endothelium levels. SLIT3 belongs to a conserved CD31hiEMCNhi endothelium3. However, it is currently unclear family of three SLIT ligands that were initially discovered in the whether and how levels of CD31hiEMCNhi endothelium are cou- context of central nervous system development, where they mediate pled with the physiological need for bone formation and whether axonal guidance through the Roundabout (ROBO) family of recep- osteoblasts participate in this regulation. tors (ROBO1–ROBO4)6,7. Subsequent studies showed that SLITs Previously, we demonstrated that the adaptor protein Schnurri3 are widely expressed, and the SLIT–ROBO pathway has been impli- (SHN3) is a suppressor of osteoblast activity, because mice lacking cated in multiple physiological functions outside of the nervous sys- SHN3 display a progressive increase in postnatal bone mass because tem, such as angiogenesis/vasculogenesis8–10, stem cell regulation11,12 of augmented bone formation4,5. Because deletion of Shn3 in osteo- and cancer development13. In addition, SLIT3 has been identified blast-lineage cells is sufficient to greatly enhance bone formation, it as a proangiogenic factor in mouse models and human engineered appears that osteoblasts are able to coordinate all of the various tis- tissue14,15. However, the role of SLIT3 in bone metabolism is still sue activities beyond osteoblast-mediated matrix secretion needed largely unclear. 1Department of Pathology and Laboratory Medicine, Cornell University, New York, NY, USA. 2Department of Orthopaedics, Shanghai Key Laboratory of Orthopaedic Implant, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. 3Laboratory of Brain Development and Repair, The Rockefeller University, New York, NY, USA. 4Division of Rheumatology, Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA. 5Research Division, Hospital for Special Surgery, New York, NY, USA. 6Department of Joint Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, China. 7Division of Adult Reconstruction and Joint Replacement, Department of Orthopaedic Surgery, Hospital for Special Surgery, New York, NY, USA. 8Institute for Computational Biomedicine, Cornell University, New York, NY, USA. 9Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Harvard University Medical School, Boston, MA, USA. 10Department of Medicine, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, USA. 11Division of Regenerative Medicine, Department of Medicine, Ansary Stem Cell Institute, Cornell University, New York, NY, USA. 12Department of Biomechanics, Hospital for Special Surgery, New York, NY, USA. 13Arthritis and Tissue Degeneration Program and David Z. Rosensweig Genomics Research Center, Hospital for Special Surgery, New York, NY, USA. 14Department of Medicine, Weill Cornell Medical College, Cornell University, New York, NY, USA. *e-mail: [email protected]; [email protected] NatURE MEDICINE | VOL 24 | JUNE 2018 | 823–833 | www.nature.com/naturemedicine 823 © 2018 Nature America Inc., part of Springer Nature. All rights reserved. ARTICLES NATURE MEDICINE Results (Fig. 2a,b and Supplementary Fig. 2a–c). On the basis of this, we SHN3 acts in osteoblasts to regulate CD31hiEMCNhi endothelium. selected the Shn3dmp1 strain for further study because it implicates To address our hypothesis that osteoblasts are able to coordinate a more restricted subpopulation in any phenotypes observed16,17. levels of osteogenic CD31hiEMCNhi endothelium to maintain bone Histomorphometric analysis confirmed that the high bone mass formation capacity, we assessed CD31hiEMCNhi endothelium levels phenotype of Shn3dmp1 mice was due to augmented osteoblast- in a mouse strain displaying augmented postnatal bone formation, mediated bone formation predominantly on the endosteal sur- namely, Shn3−/− mice. We found that CD31 and EMCN double-pos- face (Fig. 2c,d and Supplementary Fig. 2d,e). Analysis of skeletal itive endothelium was present in the marrow immediately beneath CD31hiEMCNhi endothelium by both immunostaining and flow the growth plate and was considerably higher in Shn3−/− mice than cytometry demonstrated that Shn3dmp1 mice displayed higher levels in wild-type (WT) controls. This effect was bone specific because, of this subset of endothelial cells relative to littermate Shn3f/f con- relative to littermate controls, Shn3−/− mice displayed normal levels trols in a similar manner as seen in Shn3−/− mice (Fig. 2e–g and of EMCN+ vessels in other organs, including heart, brain, lung and Supplementary Fig. 2f–h). kidney (Supplementary Fig. 1a). This vessel phenotype was pres- To further confirm that the function of SHN3 to regulate skel- ent in neonatal Shn3−/− mice and thus preceded the appearance etal CD31hiEMCNhi vascular endothelium maps to osteoblasts, we of the high bone mass phenotype5 (Supplementary Fig. 1b). Flow generated endothelial cell–specific Shn3-deficient mice (Shn3cdh5 cytometry also confirmed greater amounts of CD31hiEMCNhi vas- mice) using the Cdh5 (VE-cadherin)-Cre. Despite observing effi- cular endothelium in the bones of Shn3−/− mice than in WT litter- cient deletion of Shn3 in BM endothelial cells (Supplementary mate controls (Fig. 1e,f and Supplementary Fig. 1c). Taken together, Fig. 2i), levels of CD31hiEMCNhi endothelial cells were unchanged in these observations suggest that SHN3 regulates levels of skeletal Shn3cdh5 mice relative to littermate Shn3f/f controls (Supplementary CD31hiEMCNhi endothelium in addition to its role in regulating Fig. 2j–m). Accordingly, Shn3cdh5 mice also displayed normal bone osteoblast activity. mass relative to littermate Shn3f/f controls (Supplementary Fig. 2n). Because SHN3 acts in a cell-intrinsic manner to regulate bone Taken together with the Shn3−/− and Shn3dmp1 phenotype, we con- formation by osteoblasts, we reasoned that SHN3 also acts in osteo- clude that osteoblasts regulate skeletal CD31hiEMCNhi vascular blasts to control levels of CD31hiEMCNhi endothelium. To test this endothelium in an SHN3-dependent manner and that SHN3 does directly, we bred Shn3f/f mice to a cre-deleter strain targeting osteo- not act directly in endothelial cells to regulate skeletal phenotypes. blast progenitors, OSX-cre, and to a cre-deleter strain targeting To determine whether osteoblasts continuously participate in this mature osteoblasts, DMP1-cre. Both Shn3osx and Shn3dmp1 male mice regulation