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

Nature volume 507, pages 323–328 (2014)Cite this article

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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.

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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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Author notes

  1. Anjali P. Kusumbe and Saravana K. Ramasamy: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, D-48149 Münster, Germany,

    Anjali P. Kusumbe, Saravana K. Ramasamy & Ralf H. Adams

  2. University of Münster, Faculty of Medicine, D-48149 Münster, Germany,

    Ralf H. Adams

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  1. Anjali P. Kusumbe
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Contributions

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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