Epigenetic memory of coronavirus infection in innate immune cells and their progenitors.

Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.

A vertebral skeletal stem cell lineage driving metastasis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.

A multi-stem cell basis for craniosynostosis and calvarial mineralization.

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC1 (CTSK+ CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2+ CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans2,3, solely in CTSK+ CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK+ CSCs and a corresponding expansion of DDR2+ CSCs, with DDR2+ CSC expansion being a direct maladaptive response to CTSK+ CSC depletion. DDR2+ CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2+ CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2+ CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK+ CSCs. Finally, the human counterparts of DDR2+ CSCs and CTSK+ CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency.

Discovery of a periosteal stem cell mediating intramembranous bone formation.

Bone consists of separate inner endosteal and outer periosteal compartments, each with distinct contributions to bone physiology and each maintaining separate pools of cells owing to physical separation by the bone cortex. The skeletal stem cell that gives rise to endosteal osteoblasts has been extensively studied; however, the identity of periosteal stem cells remains unclear1-5. Here we identify a periosteal stem cell (PSC) that is present in the long bones and calvarium of mice, displays clonal multipotency and self-renewal, and sits at the apex of a differentiation hierarchy. Single-cell and bulk transcriptional profiling show that PSCs display transcriptional signatures that are distinct from those of other skeletal stem cells and mature mesenchymal cells. Whereas other skeletal stem cells form bone via an initial cartilage template using the endochondral pathway4, PSCs form bone via a direct intramembranous route, providing a cellular basis for the divergence between intramembranous versus endochondral developmental pathways. However, there is plasticity in this division, as PSCs acquire endochondral bone formation capacity in response to injury. Genetic blockade of the ability of PSCs to give rise to bone-forming osteoblasts results in selective impairments in cortical bone architecture and defects in fracture healing. A cell analogous to mouse PSCs is present in the human periosteum, raising the possibility that PSCs are attractive targets for drug and cellular therapy for skeletal disorders. The identification of PSCs provides evidence that bone contains multiple pools of stem cells, each with distinct physiologic functions.

Characterization of a novel Hoxa5eGFP mouse line.

BACKGROUND: Hox genes encode transcription factors that are important for establishing the body plan. Hoxa5 is a member of the mammalian Hox5 paralogous group that regulates the patterning and morphology of the cervical-thoracic region of the axial skeleton. Hoxa5 also plays crucial functions in lung morphogenesis. RESULTS: We generated a Hoxa5eGFP reporter mouse line using CRISPR technology, allowing real-time visualization of Hoxa5 expression. Hoxa5eGFP recapitulates reported embryonic Hoxa5 mRNA expression patterns. Specifically, Hoxa5eGFP can be visualized in the developing mouse neural tube, somites, lung, diaphragm, foregut, and midgut, among other organs. In the stomach, posteriorly biased Hoxa5eGFP expression correlates with a drastic morphological reduction of the corpus in Hox5 paralogous mutants. Expression of Hoxa5eGFP in the lung continues in all lung fibroblast populations through postnatal and adult stages. CONCLUSIONS: We identified cell types that express Hoxa5 in postnatal and adult mouse lungs, including various fibroblasts and vascular endothelial cells. This reporter line will be a powerful tool for studies of the function of Hoxa5 during mouse development, homeostasis, and disease processes.

Development of Murine Anterior Interbody and Posterolateral Spinal Fusion Techniques.

BACKGROUND: Multiple animal models have previously been utilized to investigate anterior fusion techniques, but a mouse model has yet to be developed. The purpose of this study was to develop murine anterior interbody and posterolateral fusion techniques. METHODS: Mice underwent either anterior interbody or posterolateral spinal fusion. A protocol was developed for both procedures, including a description of the relevant anatomy. Samples were subjected to micro-computed tomography to assess fusion success and underwent biomechanical testing with use of 4-point bending. Lastly, samples were fixed and embedded for histologic evaluation. RESULTS: Surgical techniques for anterior interbody and posterolateral fusion were developed. The fusion rate was 83.3% in the anterior interbody model and 100% in the posterolateral model. Compared with a control, the posterolateral model exhibited a greater elastic modulus. Histologic analysis demonstrated endochondral ossification between bridging segments, further confirming the fusion efficacy in both models. CONCLUSIONS: The murine anterior interbody and posterolateral fusion models are efficacious and provide an ideal platform for studying the molecular and cellular mechanisms mediating spinal fusion. CLINICAL RELEVANCE: Given the extensive genetic tools available in murine disease models, use of fusion models such as ours can enable determination of the underlying genetic pathways involved in spinal fusion.

Hox11 genes establish synovial joint organization and phylogenetic characteristics in developing mouse zeugopod skeletal elements.

Hox11 genes are essential for zeugopod skeletal element development but their roles in synovial joint formation remain largely unknown. Here, we show that the elbow and knee joints of mouse embryos lacking all Hox11 paralogous genes are specifically remodeled and reorganized. The proximal ends of developing mutant ulna and radius elements became morphologically similar and formed an anatomically distinct elbow joint. The mutant ulna lacked the olecranon that normally attaches to the triceps brachii muscle tendon and connects the humerus to the ulna. In its place, an ulnar patella-like element developed that expressed lubricin on its ventral side facing the joint and was connected to the triceps muscle tendon. In mutant knees, both tibia and fibula fully articulated with an enlarged femoral epiphyseal end that accommodated both elements, and the neo-tripartite knee joint was enclosed in a single synovial cavity and displayed an additional anterior ligament. The mutant joints also exhibited a different organization of the superficial zone of articular cartilage that normally exerts an anti-friction function. In conclusion, Hox11 genes co-regulate and coordinate the development of zeugopod skeletal elements and adjacent elbow and knee joints, and dictate joint identity, morphogenesis and anatomical and functional organization. Notably, the ulnar patella and tripartite knee joints in the mouse mutants actually characterize several lower vertebrates, including certain reptiles and amphibians. The re-emergence of such anatomical structures suggests that their genetic blueprint is still present in the mouse genome but is normally modified to the needs of the mammalian joint-formation program by distinct Hox11 function.

An angiogenic approach to osteoanabolic therapy targeting the SHN3-SLIT3 pathway.

Often, disorders of impaired bone formation involve not only a cell intrinsic defect in the ability of osteoblasts to form bone, but moreover a broader dysfunction of the skeletal microenvironment that limits osteoblast activity. Developing approaches to osteoanabolic therapy that not only augment osteoblast activity but moreover correct this microenvironmental dysfunction may enable both more effective osteoanabolic therapies and also addressing a broader set of indications where vasculopathy or other forms microenvironment dysfunction feature prominently. We here review evidence that SHN3 acts as a suppressor of not only the cell intrinsic bone formation activity of osteoblasts, but moreover of the creation of a local osteoanabolic microenvironment. Mice lacking Schnurri3 (SHN3, HIVEP3) display a very robust increase in bone formation, that is due to de-repression of ERK pathway signaling in osteoblasts. In addition to loss of SHN3 augmenting the differentiation and bone formation activity of osteoblasts, loss of SHN3 increases secretion of SLIT3 by osteoblasts, which in a skeletal context acts as an angiogenic factor. Through this angiogenic activity, SLIT3 creates an osteoanabolic microenvironment, and accordingly treatment with SLIT3 can increase bone formation and enhance fracture healing. These features both validate vascular endothelial cells as a therapeutic target for disorders of low bone mass alongside the traditionally targeted osteoblasts and osteoclasts and indicate that targeting the SHN3/SLIT3 pathway provides a new mechanism to induce therapeutic osteoanabolic responses.

Identification of a stem cell mediating osteoblast versus adipocyte lineage selection.

Most skeletal fragility disorders are characterized by bone loss with a concurrent gain in marrow adipocytes 1-8. This suggests that a cell that forms adipocytes at the expense of osteoblasts is central to the pathogenesis of skeletal disorders. However, this cellular point of bifurcation between adipocyte and osteoblast differentiation pathways remains unknown. Here, we identify a new cell type defined by co-expression of skeletal stem cell and adipocyte precursor markers, 9-13 (CD24+CD29+ skeletal stem cells (SSCs)), that serves as a key cellular point of bifurcation between the osteoblast and adipocyte differentiation pathways, giving rise to closely related osteoblast and adipocyte lineage-restricted precursors. CD24+CD29+SSCs comprise a small fraction of SSCs, and only this fraction displays full stemness features, including the ability to undergo serial transplantation. In line with serving as the osteoblast/adipocyte bipotent cell, the "bone to fat" tissue remodeling occurring in models of postmenopausal osteoporosis or after high fat diet exposure occur in part by reprogramming these CD24+CD29+SSCs to change their output of lineage-restricted precursors. Lastly, as subcutaneous white adipose tissue displays a similar set of CD24+CD29+ stem cells and related lineage-restricted progenitors, these findings provide a new schema explaining the stem cell basis of bone versus adipose tissue production that unifies multiple mesenchymal tissues.

Schnurri-3 inhibition rescues skeletal fragility and vascular skeletal stem cell niche pathology in a mouse model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a disorder of low bone mass and increased fracture risk due to a range of genetic variants that prominently include mutations in genes encoding type collagen. While it is well known that OI reflects defects in the activity of bone-forming osteoblasts, it is currently unclear whether OI also reflects defects in the many other cell types comprising bone, including defects in skeletal vascular endothelium or the skeletal stem cell populations that give rise to osteoblasts and whether correcting these broader defects could have therapeutic utility. Here, we find that numbers of skeletal stem cells (SSCs) and skeletal arterial endothelial cells (AECs) are augmented in Col1a2oim/oim mice, a well-studied animal model of moderate to severe OI, suggesting that disruption of a vascular SSC niche is a feature of OI pathogenesis. Moreover, crossing Col1a2oim/oim mice to mice lacking a negative regulator of skeletal angiogenesis and bone formation, Schnurri 3 (SHN3), not only corrected the SSC and AEC phenotypes but moreover robustly corrected the bone mass and spontaneous fracture phenotypes. As this finding suggested a strong therapeutic utility of SHN3 inhibition for the treatment of OI, a bone-targeting AAV was used to mediate Shn3 knockdown, rescuing the Col1a2oim/oim phenotype and providing therapeutic proof-of-concept for targeting SHN3 for the treatment of OI. Overall, this work both provides proof-of-concept for inhibition of the SHN3 pathway and more broadly addressing defects in the stem/osteoprogentior niche as is a strategy to treat OI.

Discovery of a Vertebral Skeletal Stem Cell Driving Spinal Metastases.

Vertebral bone is subject to a distinct set of disease processes from those of long bones, notably including a much higher rate of solid tumor metastases that cannot be explained by passive blood flow distribution alone. The basis for this distinct biology of vertebral bone has remained elusive. Here we identify a vertebral skeletal stem cell (vSSC), co-expressing the transcription factors ZIC1 and PAX1 together with additional cell surface markers, whose expression profile and function are markedly distinct from those of long bone skeletal stem cells (lbSSCs). vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. Lineage tracing of vSSCs confirms that they make a persistent contribution to multiple mature cell lineages in the native vertebrae. vSSCs are physiologic mediators of spine mineralization, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed clinically in breast cancer. Specifically, when an organoid system is used to place both vSSCs and lbSSCs in an identical anatomic context, vSSC-lineage cells are more efficient than lbSSC-lineage cells at recruiting metastases, a phenotype that is due in part to increased secretion of the novel metastatic trophic factor MFGE8. Similarly, genetically targeting loss-of-function to the vSSC lineage results in reduced metastasis rates in the native vertebral environment. Taken together, vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of metastatic seeding of the vertebrae.

SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts.

Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation.

Non-homeodomain regions of Hox proteins mediate activation versus repression of Six2 via a single enhancer site in vivo.

Hox genes control many developmental events along the AP axis, but few target genes have been identified. Whether target genes are activated or repressed, what enhancer elements are required for regulation, and how different domains of the Hox proteins contribute to regulatory specificity are poorly understood. Six2 is genetically downstream of both the Hox11 paralogous genes in the developing mammalian kidney and Hoxa2 in branchial arch and facial mesenchyme. Loss-of-function of Hox11 leads to loss of Six2 expression and loss-of-function of Hoxa2 leads to expanded Six2 expression. Herein we demonstrate that a single enhancer site upstream of the Six2 coding sequence is responsible for both activation by Hox11 proteins in the kidney and repression by Hoxa2 in the branchial arch and facial mesenchyme in vivo. DNA-binding activity is required for both activation and repression, but differential activity is not controlled by differences in the homeodomains. Rather, protein domains N- and C-terminal to the homeodomain confer activation versus repression activity. These data support a model in which the DNA-binding specificity of Hox proteins in vivo may be similar, consistent with accumulated in vitro data, and that unique functions result mainly from differential interactions mediated by non-homeodomain regions of Hox proteins.

A Hox-Eya-Pax complex regulates early kidney developmental gene expression.

During embryonic development, the anterior-posterior body axis is specified in part by the combinatorial activities of Hox genes. Given the poor DNA binding specificity of Hox proteins, their interaction with cofactors to regulate target genes is critical. However, few regulatory partners or downstream target genes have been identified. Herein, we demonstrate that Hox11 paralogous proteins form a complex with Pax2 and Eya1 to directly activate expression of Six2 and Gdnf in the metanephric mesenchyme. We have identified the binding site within the Six2 enhancer necessary for Hox11-Eya1-Pax2-mediated activation and demonstrate that this site is essential for Six2 expression in vivo. Furthermore, genetic interactions between Hox11 and Eya1 are consistent with their participation in the same pathway. Thus, anterior-posterior-patterning Hox proteins interact with Pax2 and Eya1, factors important for nephrogenic mesoderm specification, to directly regulate the activation of downstream target genes during early kidney development.

MEKK2 mediates aberrant ERK activation in neurofibromatosis type I.

Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1fl/fl;Dmp1-Cre) and Mekk2-/- each displaying skeletal defects, Nf1fl/fl;Mekk2-/-;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.

Histone demethylase LSD1 is critical for endochondral ossification during bone fracture healing.

Bone fracture is repaired predominantly through endochondral ossification. However, the regulation of endochondral ossification by key factors during fracture healing remains largely enigmatic. Here, we identify histone modification enzyme LSD1 as a critical factor regulating endochondral ossification during bone regeneration. Loss of LSD1 in Prx1 lineage cells severely impaired bone fracture healing. Mechanistically, LSD1 tightly controls retinoic acid signaling through regulation of Aldh1a2 expression level. The increased retinoic acid signaling in LSD1-deficient mice suppressed SOX9 expression and impeded the cartilaginous callus formation during fracture repair. The discovery that LSD1 can regulate endochondral ossification during fracture healing will benefit the understanding of bone regeneration and have implications for regenerative medicine.

Osteoclasts are not a source of SLIT3.

The axon guidance cue SLIT3 was identified as an osteoanabolic agent in two recent reports. However, these reports conflict in their nomination of osteoblasts versus osteoclasts as the key producers of skeletal SLIT3 and additionally offer conflicting data on the effects of SLIT3 on osteoclastogenesis. Here, aiming to address this discrepancy, we found no observable SLIT3 expression during human or mouse osteoclastogenesis and the only modest SLIT3-mediated effects on osteoclast differentiation. Conditional deletion of SLIT3 in cathepsin K (CTSK)-positive cells, including osteoclasts, had no effect on the number of osteoclast progenitors, in vitro osteoclast differentiation, overall bone mass, or bone resorption/formation parameters. Similar results were observed with the deletion of SLIT3 in LysM-positive cells, including osteoclast lineage cells. Consistent with this finding, bone marrow chimeras made from Slit3 -/- donors that lacked SLIT3 expression at all stages of osteoclast development displayed normal bone mass relative to controls. Taken in context, multiple lines of evidence were unable to identify the physiologic function of osteoclast-derived SLIT3, indicating that osteoblasts are the major source of skeletal SLIT3.

Irradiation induces p53 loss of heterozygosity in breast cancer expressing mutant p53.

Mutations in one allele of the TP53 gene in cancer early stages are frequently followed by the loss of the remaining wild-type allele (LOH) during tumor progression. However, the clinical impact of TP53 mutations and p53LOH, especially in the context of genotoxic modalities, remains unclear. Using MMTV;ErbB2 model carrying a heterozygous R172H p53 mutation, we report a previously unidentified oncogenic activity of mutant p53 (mutp53): the exacerbation of p53LOH after irradiation. We show that wild-type p53 allele is partially transcriptionally competent and enables the maintenance of the genomic integrity under normal conditions in mutp53 heterozygous cells. In heterozygous cells γ-irradiation promotes mutp53 stabilization, which suppresses DNA repair and the cell cycle checkpoint allowing cell cycle progression in the presence of inefficiently repaired DNA, consequently increases genomic instability leading to p53LOH. Hence, in mutp53 heterozygous cells, irradiation facilitates the selective pressure for p53LOH that enhances cancer cell fitness and provides the genetic plasticity for acquiring metastatic properties.

Heat shock factor 1 confers resistance to lapatinib in ERBB2-positive breast cancer cells.

Despite success of ERBB2-targeted therapies such as lapatinib, resistance remains a major clinical concern. Multiple compensatory receptor tyrosine kinase (RTK) pathways are known to contribute to lapatinib resistance. The heterogeneity of these adaptive responses is a significant hurdle for finding most effective combinatorial treatments. The goal of this study was to identify a unifying molecular mechanism whose targeting could help prevent and/or overcome lapatinib resistance. Using the MMTV-ERBB2;mutant p53 (R175H) in vivo mouse model of ERBB2-positive breast cancer, together with mouse and human cell lines, we compared lapatinib-resistant vs. lapatinib-sensitive tumor cells biochemically and by kinome arrays and evaluated their viability in response to a variety of compounds affecting heat shock response. We found that multiple adaptive RTKs are activated in lapatinib-resistant cells in vivo, some of which have been previously described (Axl, MET) and some were novel (PDGFRα, PDGFRß, VEGFR1, MUSK, NFGR). Strikingly, all lapatinib-resistant cells show chronically activated HSF1 and its transcriptional targets, heat shock proteins (HSPs), and, as a result, superior tolerance to proteotoxic stress. Importantly, lapatinib-resistant tumors and cells retained sensitivity to Hsp90 and HSF1 inhibitors, both in vitro and in vivo, thus providing a unifying and actionable therapeutic node. Indeed, HSF1 inhibition simultaneously downregulated ERBB2, adaptive RTKs and mutant p53, and its combination with lapatinib prevented development of lapatinib resistance in vitro. Thus, the kinome adaptation in lapatinib-resistant ERBB2-positive breast cancer cells is governed, at least in part, by HSF1-mediated heat shock pathway, providing a novel potential intervention strategy to combat resistance.

Targeting skeletal endothelium to ameliorate bone loss.

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 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.