Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone

  • A Corrigendum to this article was published on 24 September 2014

Abstract

The mammalian skeletal system harbours a hierarchical system of mesenchymal stem cells, osteoprogenitors and osteoblasts sustaining lifelong bone formation. Osteogenesis is indispensable for the homeostatic renewal of bone as well as regenerative fracture healing, but these processes frequently decline in ageing organisms, leading to loss of bone mass and increased fracture incidence. Evidence indicates that the growth of blood vessels in bone and osteogenesis are coupled, but relatively little is known about the underlying cellular and molecular mechanisms. Here we identify a new capillary subtype in the murine skeletal system with distinct morphological, molecular and functional properties. These vessels are found in specific locations, mediate growth of the bone vasculature, generate distinct metabolic and molecular microenvironments, maintain perivascular osteoprogenitors and couple angiogenesis to osteogenesis. The abundance of these vessels and associated osteoprogenitors was strongly reduced in bone from aged animals, and pharmacological reversal of this decline allowed the restoration of bone mass.

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Figure 1: Identification of bone vessel subtypes.
Figure 2: Osteoprogenitor association with type H endothelial cells.
Figure 3: Properties and age-dependent decline of type H endothelial cells.
Figure 4: Type H endothelial cells mediate bone vessel growth.
Figure 5: Type H endothelial cells couple angiogenesis and osteogenesis.

References

  1. 1

    Tashiro, Y. et al. Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice. Blood 119, 6382–6393 (2012)

    CAS  Google Scholar 

  2. 2

    Red-Horse, K., Crawford, Y., Shojaei, F. & Ferrara, N. Endothelium-microenvironment interactions in the developing embryo and in the adult. Dev. Cell 12, 181–194 (2007)

    CAS  Google Scholar 

  3. 3

    Ding, L., Saunders, T. L., Enikolopov, G. & Morrison, S. J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012)

    CAS  ADS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Butler, J. M., Kobayashi, H. & Rafii, S. Instructive role of the vascular niche in promoting tumour growth and tissue repair by angiocrine factors. Nature Rev. Cancer 10, 138–146 (2010)

    CAS  Google Scholar 

  5. 5

    Ribatti, D., Nico, B., Vacca, A., Roncali, L. & Dammacco, F. Endothelial cell heterogeneity and organ specificity. J. Hematother. Stem Cell Res. 11, 81–90 (2002)

    Google Scholar 

  6. 6

    Garlanda, C. & Dejana, E. Heterogeneity of endothelial cells. Specific markers. Arterioscler. Thromb. Vasc. Biol. 17, 1193–1202 (1997)

    CAS  Google Scholar 

  7. 7

    Eshkar-Oren, I. et al. The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development 136, 1263–1272 (2009)

    CAS  Google Scholar 

  8. 8

    Maes, C. et al. Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones. EMBO J. 29, 424–441 (2010)

    CAS  Google Scholar 

  9. 9

    Trueta, J. & Buhr, A. J. The vascular contribution to osteogenesis. V. the vasculature supplying the epiphysial cartilage in rachitic rats. J. Bone Joint Surg. Br. 45, 572–581 (1963)

    CAS  Google Scholar 

  10. 10

    Trueta, J. & Morgan, J. D. The vascular contribution to osteogenesis. I. Studies by the injection method. J. Bone Joint Surg. Br. 42-B, 97–109 (1960)

    CAS  Google Scholar 

  11. 11

    Glowacki, J. Angiogenesis in fracture repair. Clin. Orthop. Relat. Res. 355, (suppl.),. S82–S89 (1998)

    Google Scholar 

  12. 12

    Maes, C. et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev. Cell 19, 329–344 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Burkhardt, R. et al. Changes in trabecular bone, hematopoiesis and bone marrow vessels in aplastic anemia, primary osteoporosis, and old age: a comparative histomorphometric study. Bone 8, 157–164 (1987)

    CAS  Google Scholar 

  14. 14

    Lu, C. et al. Effect of age on vascularization during fracture repair. J. Orthop. Res. 26, 1384–1389 (2008)

    PubMed  PubMed Central  Google Scholar 

  15. 15

    Kataoka, M. & Tavassoli, M. Identification of lectin-like substances recognizing galactosyl residues of glycoconjugates on the plasma membrane of marrow sinus endothelium. Blood 65, 1163–1171 (1985)

    CAS  Google Scholar 

  16. 16

    Kopp, H. G., Avecilla, S. T., Hooper, A. T. & Rafii, S. The bone marrow vascular niche: home of HSC differentiation and mobilization. Physiology (Bethesda) 20, 349–356 (2005)

    CAS  Google Scholar 

  17. 17

    Hooper, A. T. et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell 4, 263–274 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Nombela-Arrieta, C. et al. Quantitative imaging of haematopoietic stem and progenitor cell localization and hypoxic status in the bone marrow microenvironment. Nature Cell Biol. 15, 533–543 (2013)

    CAS  Google Scholar 

  19. 19

    Trueta, J. & Harrison, M. H. The normal vascular anatomy of the femoral head in adult man. J. Bone Joint Surg. Br. 35-B, 442–461 (1953)

    CAS  Google Scholar 

  20. 20

    Crock, H. V. A revision of the anatomy of the arteries supplying the upper end of the human femur. J. Anat. 99, 77–88 (1965)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Ullah, M. S., Davies, A. J. & Halestrap, A. P. The plasma membrane lactate transporter MCT4, but not MCT1, is up-regulated by hypoxia through a HIF-1α-dependent mechanism. J. Biol. Chem. 281, 9030–9037 (2006)

    CAS  PubMed  Google Scholar 

  22. 22

    Boado, R. J. & Pardridge, W. M. Glucose deprivation and hypoxia increase the expression of the GLUT1 glucose transporter via a specific mRNA cis-acting regulatory element. J. Neurochem. 80, 552–554 (2002)

    CAS  Google Scholar 

  23. 23

    Rodríguez, J. et al. ERK1/2 MAP kinases promote cell cycle entry by rapid, kinase-independent disruption of retinoblastoma-lamin A complexes. J. Cell Biol. 191, 967–979 (2010)

    PubMed  PubMed Central  Google Scholar 

  24. 24

    Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010)

    CAS  Article  ADS  Google Scholar 

  25. 25

    Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007)

    CAS  PubMed  Google Scholar 

  26. 26

    Skawina, A., Litwin, J. A., Gorczyca, J. & Miodonski, A. J. The vascular system of human fetal long bones: a scanning electron microscope study of corrosion casts. J. Anat. 185, 369–376 (1994)

    PubMed  PubMed Central  Google Scholar 

  27. 27

    Nakashima, K. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 (2002)

    CAS  PubMed  Google Scholar 

  28. 28

    Lips, P., Courpron, P. & Meunier, P. J. Mean wall thickness of trabecular bone packets in the human iliac crest: changes with age. Calcif. Tissue Res. 26, 13–17 (1978)

    CAS  Google Scholar 

  29. 29

    Smith, D. M., Khairi, M. R. & Johnston, C. C., Jr The loss of bone mineral with aging and its relationship to risk of fracture. J. Clin. Invest. 56, 311–318 (1975)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Šale, S., Lafkas, D. & Artavanis-Tsakonas, S. Notch2 genetic fate mapping reveals two previously unrecognized mammary epithelial lineages. Nature Cell Biol. 15, 451–460 (2013)

    Google Scholar 

  31. 31

    Zovein, A. C. et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell 3, 625–636 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Swift, M. R. & Weinstein, B. M. Arterial-venous specification during development. Circ. Res. 104, 576–588 (2009)

    CAS  Google Scholar 

  33. 33

    Helisch, A. & Schaper, W. Arteriogenesis: the development and growth of collateral arteries. Microcirculation 10, 83–97 (2003)

    Google Scholar 

  34. 34

    Pugh, C. W. & Ratcliffe, P. J. Regulation of angiogenesis by hypoxia: role of the HIF system. Nature Med. 9, 677–684 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Tang, N. et al. Loss of HIF-1α in endothelial cells disrupts a hypoxia-driven VEGF autocrine loop necessary for tumorigenesis. Cancer Cell 6, 485–495 (2004)

    CAS  Google Scholar 

  36. 36

    Tanimoto, K., Makino, Y., Pereira, T. & Poellinger, L. Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor suppressor protein. EMBO J. 19, 4298–4309 (2000)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Jones, D. T. & Harris, A. L. Identification of novel small-molecule inhibitors of hypoxia-inducible factor-1 transactivation and DNA binding. Mol. Cancer Ther. 5, 2193–2202 (2006)

    CAS  Google Scholar 

  38. 38

    Kuznetsov, S. A. et al. The interplay of osteogenesis and hematopoiesis: expression of a constitutively active PTH/PTHrP receptor in osteogenic cells perturbs the establishment of hematopoiesis in bone and of skeletal stem cells in the bone marrow. J. Cell Biol. 167, 1113–1122 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Bianco, P. Bone and the hematopoietic niche: a tale of two stem cells. Blood 117, 5281–5288 (2011)

    CAS  Google Scholar 

  40. 40

    Long, M. W. Osteogenesis and bone-marrow-derived cells. Blood Cells Mol. Dis. 27, 677–690 (2001)

    CAS  Google Scholar 

  41. 41

    Yin, T. & Li, L. The stem cell niches in bone. J. Clin. Invest. 116, 1195–1201 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Shapiro, F. Bone development and its relation to fracture repair. The role of mesenchymal osteoblasts and surface osteoblasts. Eur. Cell. Mater. 15, 53–76 (2008)

    CAS  Google Scholar 

  43. 43

    Park, D. et al. Endogenous bone marrow MSCs are dynamic, fate-restricted participants in bone maintenance and regeneration. Cell Stem Cell 10, 259–272 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Ellis, S. L. et al. The relationship between bone, hemopoietic stem cells, and vasculature. Blood 118, 1516–1524 (2011)

    CAS  Google Scholar 

  45. 45

    Parmar, K., Mauch, P., Vergilio, J. A., Sackstein, R. & Down, J. D. Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc. Natl Acad. Sci. USA 104, 5431–5436 (2007)

    CAS  ADS  Google Scholar 

  46. 46

    Wang, L. et al. Identification of a clonally expanding haematopoietic compartment in bone marrow. EMBO J. 32, 219–230 (2013)

    Google Scholar 

  47. 47

    Ramasamy, S. K., Kusumbe, A. P. & Adams, R. H. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature http://dx.doi.org/10.1038/nature13146 (this issue)

  48. 48

    Heaney, R. P. Pathophysiology of osteoporosis. Endocrinol. Metab. Clin. North Am. 27, 255–265 (1998)

    CAS  Google Scholar 

  49. 49

    Losordo, D. W. & Isner, J. M. Estrogen and angiogenesis: A review. Arterioscler. Thromb. Vasc. Biol. 21, 6–12 (2001)

    CAS  Google Scholar 

  50. 50

    Haase, V. H., Glickman, J. N., Socolovsky, M. & Jaenisch, R. Vascular tumors in livers with targeted inactivation of the von Hippel-Lindau tumor suppressor. Proc. Natl Acad. Sci. USA 98, 1583–1588 (2001)

    CAS  ADS  Google Scholar 

  51. 51

    Xu, Y. et al. Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3. J. Cell Biol. 188, 115–130 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Medvinsky for kindly providing Flk1-GFP mice, M. Stehling for endothelial cell sorting and A. Borgscheiper for technical assistance. Funding was provided by the Max Planck Society, the University of Münster, the DFG cluster of excellence ‘Cells in Motion’ and the European Research Council (AdG 339409 AngioBone).

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A.P.K., S.K.R. and R.H.A. designed experiments and interpreted results. A.P.K and S.K.R performed all experiments. A.P.K and R.H.A. wrote the manuscript.

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Correspondence to Ralf H. Adams.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schematic representation of key findings.

a, Type H vasculature (red) in the metaphysis (mp) and endosteum (es) represents a functionally specialized vessel subtype that mediates vessel growth and promotes osteogenesis. The later is presumably mediated by angiocrine growth factors (small circles). In aged animals (right), the number of type H vessels and associated osteoprogenitors (OPs) is strongly reduced so that bone mainly contains type L, sinusoidal vessels characteristic for the diaphyseal (dp) marrow cavity. Arrows indicate the incoming arterial flow and venous drainage. b, Regulation of type H endothelium by hypoxia-inducible factor. Endothelial-cell-specific gene inactivation of HIF-1α led to pronounced reduction of type H endothelial cells and osteoprogenitors, whereas the opposite effect was obtained by disrupting endothelial VHL expression. Type H endothelial cells, associated osteoprogenitor cells and bone formation were stimulated by DFM in aged mice.

Extended Data Figure 2 Regional differences in metabolic marker expression.

a, Tile scan confocal images showing maximum intensity surface projection of Emcn (red) immunostaining on tibial bone section. Nuclei in left image are stained with DAPI (blue). Arrowheads mark the exit of the vein through the cortical bone (cb). Indicated are growth plate (gp), diaphysis (dp), metaphysis (mp) and chondrocytes (ch). b, Schematic representation of proximal tibial bone indicating localization of different regions: secondary ossification centre (soc), growth plate (gp), metaphysis (mp), diaphysis (dp), endosteum (es). c, Representative confocal images showing pimonidazole (green) staining on a tibial section from a 5-week-old mouse. Nuclei, DAPI (blue); ECs, Endomucin (red, Emcn). Note abundance of pimonidazole staining thoughout the diaphysis (dp) but not in the metaphyseal (mp) region. Dashed lines indicate the borders of cortical bone and growth plate (gp). d, e, Maximum intensity projections of pimonidazole (green) stained 8-week-old tibia. Nuclei, DAPI (blue); CD31 (red, d); CD45 (red, e). Green staining is seen in CD45+ haematopoietic cells in the diaphysis (dp) and on the bone surface (arrowheads). Dashed lines indicate the borders of cortical bone (cb) and growth plate (gp).

Extended Data Figure 3 Regional differences in metabolic marker expression.

a, Representative confocal images showing HIF1-α (green) and CD31 (red) immunostaining on sections of 7-week-old tibiae. Nuclei, DAPI (blue). Note abundance of HIF1α-positive nuclei in the diaphysis (dp) and secondary ossification centre (soc) but not in the metaphyseal (mp) region near the growth plate (gp). Dashed lines indicate borders of the growth plate (top and centre) or, in panels on the bottom, the endosteum (es). b, Maximum intensity projections of tibial sections from 7-week-old mice showing immunostaining for the indicated markers. MCT4-positive (green) cells were absent in the metaphysis (mp) but abundant in the diaphysis (dp) and secondary ossification centre (soc). c, Maximum intensity projections of Glut1 (green) immunostaining on sections of 7-week-old tibiae. Nuclei, DAPI (blue). Note abundance of Glut1-positive cells in the diaphysis (dp) but not in the metaphyseal (mp) region below the growth plate (gp). Dashed lines indicate borders of growth plate or endosteum (es), respectively. d, Quantitation of HIF1-α, MCT4 and Glut1 immunostaining intensities in metaphysis and diaphysis. Data represent mean ± s.e.m. (n = 5 mice in two independent experiments). P values, two-tailed unpaired t-test. e, Representative tile scan confocal image showing the distribution of phospho-ERK1/2 (green) immunosignal in sections of 7-week-old tibia. Nuclei, DAPI (blue). Note prominent phospho-ERK1/2 staining of cells in the metaphysis (mp) relative to the diaphysis (dp). Dashed line indicates border of the growth plate (gp).

Extended Data Figure 4 Structural and marker heterogeneity in bone sinusoidal endothelium.

a, Representative tile scan confocal image showing maximum intensity surface projection of Endomucin (red) immunostaining of ECs in the femur of a 2-week-old mouse. Differences in Endomucin staining intensity are lost in this projection. Nuclei in left image are stained with DAPI (blue). Dashed lines indicate the adjacent growth plate (top), the border of the diaphysis (dp) and the morphologically distinct metaphyseal (mp) and diaphyseal (dp) vessels. b, Tile scan confocal image showing maximum intensity surface projection of GFP+ ECs in 2-week-old Flk1-GFP transgenic tibia. Left: nuclei, DAPI (blue). Dashed lines indicate the adjacent growth plate (top) or the border of the diaphysis (dp). Dashed lines mark borders of the growth plate (top) and cortical bone (left and right) as well as the interface between column-like metaphyseal (mp) vessels and the highly branched diaphyseal (dp) vasculature. c–e, Representative tile scan confocal images of the GFP+ (green) endothelium in Cdh5(PAC)-CreERT2, Rosa26-mT/mG double transgenic tibiae from 2-week-old (c), 4-week-old (d), or 6-week-old (e) mice after postnatal tamoxifen administration. Nuclei, DAPI (blue). GFP signal is restricted to vessels and absent in chondrocytes of the growth plate (gp) or haematopoietic cells. Dashed lines indicate the borders of the growth plate, cortical bone and secondary ossification centre (soc). f, Quantitative analysis of relative CD31 and Endomucin immunostaining intensities in the microvasculature of the metaphysis, diaphysis (marrow cavity) and endosteum, as indicated. Data represent mean ± s.e.m. (n = 7 mice from seven independent experiments). P values, two-tailed unpaired t-test. g, Mean fluorescence intensities (MFI) of CD31hiEmcnhi and CD31loEmcnlo endothelial subsets as determined by flow cytometric analysis of bone marrow cells stained with CD31 and Endomucin. Data represent mean ± s.e.m. (n = 7 mice two independent experiments). P values, two-tailed unpaired t-test.

Extended Data Figure 5 EC subsets in different skeletal elements and organs.

a–d, Representative tile scan confocal images showing CD31 (green) and Endomucin (red) immunostaining in juvenile (4-week-old) vertebra (a), sternum (b), and whole-mount (c) or sectioned (d) calvarium (parietal bone). Nuclei, DAPI (blue). Arrowheads indicate CD31hiEmcnhi endothelium (yellow). Arrow in a marks an adjacent artery (green). e, Representative dot plots showing flow cytometric analysis of CD31 and Endomucin-stained single cell suspensions from kidney, heart, spleen, lung, brain and liver. Note absence of a CD31hiEmcnhi EC subset (orange dashed circle in Q2) in these organs with exception of liver.

Extended Data Figure 6 Regional and age related differences in ECs and bone.

a, Confocal images showing diaphyseal region of flushed tibia immunostained for CD31 (green) and Endomucin (red). Nuclei, DAPI (blue). Note retention of type H endothelium in the endosteum after flushing. b, Comparative qPCR analysis of marrow cell suspension flushed from the tibial diaphysis and compact bone plus endosteum (harbouring type H endothelium). Shown are expression levels of Pdgfa, Pdgfb, Fgf1, Tgfb1 and Tgfb3 mRNAs relative to mRNA for β-actin. Data represent mean ± s.e.m. (n = 6–8 mice in two independent experiments). P values, two-tailed unpaired t-test. c, Representative confocal images from metaphyseal and diaphyseal regions of tibias from mice of different ages immunostained for Osterix (green) and CD31 (red). Nuclei, DAPI (blue). Dashed lines mark the adjacent growth plate (metaphysis) or endosteum (es) in diaphysis. Note striking decline of CD31+ vessels and associated osteoprogenitors in ageing mice. d, Quantitative mRNA expression analysis of Cspg4, Pdgfrb, Runx2 and Sp7 relative to transcripts encoding β-actin in long bones from juvenile and aged mice. Note significant decline of all 4 markers in bone from aged mice. Data represent mean ± s.e.m. (n = 7 mice in two independent experiments). P values, two-tailed unpaired t-test. e, Representative µ-CT images of tibias from juvenile (5-week-old) and aged mice. Note significant loss of bone in aged mice. f, Quantitative µ-CT analysis of relative bone volume (bone volume/total volume), number of trabeculae and trabecular separation (that is, space between trabeculae) in proximal tibias from juvenile mice and aged mice. Data represent mean ± s.e.m. (n = 5 mice in two independent experiments). P values, two-tailed unpaired t-test. g, FACS plots of CD31 and Endomucin double stained single cell suspensions from murine tibias. CD31hiEmcnhi ECs decline with age. h, Confocal images showing type H endothelium identified as GFP+ (red) ECs and proliferation (EdU incorporation, green) in the metaphysis (mp, upper panel) or diaphysis (dp, lower panel) from 3- or 8-week-old Flk1-GFP transgenic tibia, as indicated. EdU+ proliferating GFP+ cells (arrowheads), which represent type H endothelium, are abundant in the metaphysis (mp) of juvenile mice and but not adult mice. EdU+GFP+ ECs are sparse in sinusoidal (type L) vessels of the diaphysis. i, FACS plots showing CD31 and endomucin double staining of single cell suspensions from juvenile, adult and aged mice livers. CD31hiEmcnhi ECs in liver do not decline with age.

Extended Data Figure 7 Lineage tracing of type H endothelium and analysis of HIF pathway mutants.

a, b, Representative confocal images of sectioned tibiae from the genetic lineage tracing experiment analysed at 1 and 40 days after tamoxifen administration. GFP-labelled endothelial cells (green) seen in Cdh5(PAC)-CreERT2, Rosa26-mT/mG double transgenics after 1 day correspond arteries and type H endothelium (arrowheads) in the metaphysis (mp) and endosteum, but not in sinusoidal vessels of the diaphysis (dp). Counterstaining of ECs with anti-Endomucin antibody (red fluorescence) in samples taken after 40 days shows expansion of the GFP+ endothelium into the diaphyseal microvasculature. Nuclei in small insets in (a), DAPI (blue). Dashed lines indicate border of growth plate (gp) and outline of compact bone. c, Representative confocal images of metaphyseal (mp) region in tibiae from the genetic lineage tracing experiment analysed at day 40 after tamoxifen administration showing GFP-labelled ECs (green) in Cdh5(PAC)-CreERT2, Rosa26-mT/mG double transgenics and Osterix staining (white). Nuclei stained with DAPI (blue). Dashed lines drawn below chondrocytes (ch) lines indicate border of growth plate (gp). d, e, Quantitative analysis of CD31hiEmcnhi endothelial cells in long bone from Hif1aiΔEC and corresponding littermate controls analysed at postnatal day 20 (P20, d) or P37 (e). Shown is fold change in frequency of endothelial cells CD31hiEmcnhi ECs identified by flow cytometry. Data represent mean ± s.e.m. (n = 7 mice from three independent experiments (d); n = 6 mice from three independent experiments (e)). P values, two-tailed unpaired t-test. f, Quantitative analysis of CD31hiEmcnhi endothelial cells in long bone from VhliΔEC and corresponding littermate controls analysed at postnatal day (P20). Shown is fold change in frequency of endothelial cells CD31hiEmcnhi endothelial cells identified by flow cytometry. Data represent mean ± s.e.m. (n = 5 mice from three independent experiments). P values, two-tailed unpaired t-test. g, Representative tile scan confocal images from tibia sections of control and VhliΔEC mutants immunostained for Endomucin (red) and Osteopontin (green). Nuclei, DAPI (blue). Note widespread osteopontin staining in VhliΔEC mutant tibia. gp, growth plate; mp, metaphysis; dp, diaphysis; es, endosteum.

Extended Data Figure 8 VhliΔEC mutants show increased bone mass.

a, b, Serum alkaline phosphatase levels in Hif1aiΔEC (a) and VhliΔEC (b) mutants. Data represent mean ± s.e.m. (n = 5 or 6 mice for Hif1aiΔEC mice from three independent experiments; n = 6 mice for VhliΔEC from three independent experiments). P values, two-tailed unpaired t-test. c, Representative µ-CT images of tibias from VhliΔEC mutants and littermate controls. d–g, Quantitative µ-CT analysis of relative bone volume (bone volume/total volume d), trabecular thickness (e) trabecular number (f), and trabecular separation (g) in proximal tibia from VhliΔEC mutants and their littermate controls. Data represent mean ± s.e.m. (n = 4 mice from from two independent experiments). P values, two-tailed unpaired t-test. Note increased bone mass in VhliΔEC mutants. h, Calcein double labelling of 5-week-old VhliΔEC mutant and littermate control tibiae. i, j, Quantitative analysis of bone formation parameters. Mineral apposition rate (MAR; i) and bone formation rate/bone surface (BFR/BS; j) for VhliΔEC mutants and controls. Data represent mean ± s.e.m. (n = 6 or 7 mice from three independent experiments). P values, two-tailed unpaired t-test. k, Representative confocal images showing Calcitonin receptor staining (osteoclasts) in tibia sections from VhliΔEC mutants and littermate controls. Nuclei, DAPI (blue). l, m, Histomorphometric analysis of VhliΔEC and control tibiae showing osteoclast number/bone perimeter (No. Oc./B. Pm; l) and osteoclast surface/bone surface (Oc. S/B. S; m). Data represent mean ± s.e.m. (n = 6 mice from three independent experiments). P values, two-tailed unpaired t-test.

Extended Data Figure 9 Age-dependent endothelial HIF1-α expression.

a, Representative confocal images showing HIF1-α (green) and CD31 (red) immunostaining on sections of 2-week-old tibia. Nuclei, DAPI (blue). Note abundance of HIF1-α-positive type H ECs in 2-week-old metaphysis (mp) but not in the type L endothelium in diaphysis (dp). Dashed line marks border of growth plate (gp). b, Quantitative mRNA expression analysis of Hif1a transcripts relative to mRNA encoding β-actin in type H and type L. Data represent mean ± s.e.m. (n = 3 biological replicates). P values, two-tailed unpaired t-test. c, d, Maximum intensity projections of HIF1-α (green) and CD31 (red) immunostaining in 7-week-old (c) and 61-week-old (d) tibiae. Nuclei, DAPI (blue). HIF1-α-positive endothelium was not detected in metaphysis (mp) of 7-week-old (c) and 61-week-old tibia (d). e, Maximum intensity confocal images from the diaphysis of 5-week-old Flk1-GFP (green) irradiated (900 rads) and control tibiae after HIF1-α (red) immunostaining. HIF1-α signals (arrowheads) in GFP+ endothelial cells are enhanced after irradiation. f, qPCR expression analysis of Hif1a relative to transcripts encoding β-actin in FACS-isolated endothelial cells from bones of irradiated mice and untreated controls. Data represent mean ± s.e.m. (n = 7 mice from three independent experiments). P values, two-tailed unpaired t-test.

Extended Data Figure 10 DFM induction of type H endothelial cells and osteoprogenitors.

a, b, Representative confocal images of CD31 (red, a, b) or Osterix (green, b) stained tibia sections from aged DFM-treated (right) and control (left) mice (60–65-weeks-old). Low intensity projection shows only CD31hi cells. DFM induces CD31hi vessels and Osterix+ osteoprogenitors (arrowheads). Chondrocytes, ch. c, Tile-scan confocal images of CD31 (red) and Osterix (green, Osx) from metaphysis region of stained tibia sections from aged DFM-treated (right) and control (left) mice. Low intensity projection shows only CD31hi cells. DFM induces CD31hi vessels and Osterix+ osteoprogenitors (arrowheads). Nuclei, DAPI (blue). Dashed lines mark growth plate chondrocytes (ch) and outline of compact bone. Arrowheads indicate Osterix+ cells in secondary ossification centre (soc) and DFM-treated metaphysis. d, Quantitation of Osterix+ osteoprogenitor cells in DFM treated and control parietal bones. Data represent mean ± s.e.m. (n = 6 mice from mice from two independent experiments). P values, two-tailed unpaired t-test. e, qPCR analysis of Ibsp, Sp7, Bglap and mRNA expression levels relative to Actb in the aged DFM or vehicle-treated (Control) parietal bones, as indicated. Data represent mean ± s.e.m. (n = 6 or 7 mice from two independent experiments). P values, two-tailed unpaired t-test.

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Kusumbe, A., Ramasamy, S. & Adams, R. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014). https://doi.org/10.1038/nature13145

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