Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Representative FACS plots showing gating strategies for the isolation of ECs from Cdh5-mTnG reporter bone. b . Tile scan confocal images showing sections of 6, 22 or 55-week-old Cdh5-mTnG reporter bone co-stained for EMCN (red) and VEGFR3 (blue). The avascular growth plate (GP) and regions containing bmECs, mpECs and rECs are indicated. Small panels show higher magnifications of these areas. Arrowheads mark type R capillaries. Scale bars, 100μm (overview images) and 500μm (small panels). c . Heatmap showing the expression of selected marker genes for each EC subcluster (based on integrated scRNA-seq data for all age groups). d . Representative confocal images showing immunostaining for EMCN (red) and SOX11 (green, white arrowheads), marking pECs in the young but not adult metaphysis. e . CXADR (green) staining of bmECs in young, adult, and aging femur. Growth plate (GP) and epiphysis are indicated. f . MAdCAM1 (green, white arrowheads) expression in diaphyseal bmECs in young and adult femur. Scale bars, 50μm ( d ), 500μm ( e ), 100μm ( f ).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation, Staining, Expressing, Marker, Immunostaining

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b , Freshly isolated wild-type femurs at the age of 3, 6, 8 or 12 weeks (W), as indicated. Tile scan confocal images of EMCN + VEGFR3 − ECs (arrowheads) in sectioned femur at the indicated ages ( b ). Epiphysis and growth plate are labelled. Scale bars, 1000μm ( a ), 500μm ( b ). c . The UMAP plot (integrated scRNA-seq data of all age groups) showing Bmx expression in arterial endothelial cells (aECs), marked by arrowhead. Scheme of Bmx-CreERT2 genetic fate mapping. 4-hydroxy tamoxifen (4-OHT) administration (1mg/mouse) is indicated by black arrow and red arrows mark time points of analysis. d, e , High-resolution confocal images and tile scan overview images of fate-tracked Bmx-CreERT2 R26-mTmG (GFP, green) in femur at the indicated time points after 4-OHT administration. Insets in ( e ) show metaphysis (i) and diaphysis (ii), respectively. White arrowheads mark CAV1 + EMCN − aECs and yellow arrowheads GFP + CAV1 + EMCN − aECs. Green arrowheads indicate EMCN + CAV1 + rECs, which are devoid of GFP signal. Scale bars, 100μm ( d ) and 500μm or 100μm ( e ).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation, Expressing

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plots showing colour-coded subclusters in juvenile and adult bone ECs. Dashed black line encompasses rEC clusters. Red arrowhead indicates expansion of type R ECs in adult. b , Bar plots showing colour-coded subclusters in juvenile and adult bone ECs. Relative representation (in %) of rECs and aECs is indicated. c , High-magnification images of femurs immunostained for EMCN (red), VEGFR3 (blue) and CAV1 (green) showing the emergence of EMCN + VEGFR3 − CAV1 + (yellow) rECs (white arrowheads) around trabecular bone (TB). d , Two-photon microscopic image of immunostained EMCN + VEGFR3 − rECs (white arrowheads) around TB visualized by second-harmonic generation. e , Representative confocal images of 3-, 6-, 8- and 12-week-old femurs immunostained for EMCN (red) and VEGFR3 (green). White arrowheads indicate increasing age-dependent abundance of EMCN + VEGFR3 − rECs around TB. f , Quantitation of rECs (EMCN + VEGFR3 − area) showing the age-dependent increase in the 3-, 6-, 8- and 12-week-old TB relative to samples from 3 weeks. n = 3 mice per group. Mean ± s.e.m. P values, one-way analysis of variance (ANOVA). g , Schematic representation of type R capillary expansion in postnatal and adult long bone. Data in a show individual samples from different age groups, whereas b is based on integrated scRNA-seq data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Perfusion of EMCN + VEGFR3 − type R capillaries (arrowheads) near TB demonstrated by injected 2,000 kDa TRITC dextran (yellow) in 12-week-old wild-type femur. b , Representative confocal images of 12-week-old Efnb2-H2B-GFP (green) femur section co-stained for EMCN (red) and VEGFR3 (blue). Efnb2 + EMCN + rECs (white arrowheads) are connected to Efnb2 + EMCN − arterioles and arteries (green arrowheads). c , d , Type III collagen (COL3A1) ( c ) and type IV collagen (COL4A1) ( d ) are tightly associated with EMCN + VEGFR3 − type R capillaries (arrowheads) in 12-week-old wild-type femur, whereas the surrounding sinusoidal vessels show a loose reticular fibre network. e , High-magnification images showing filopodia (arrowheads) extending from EMCN + VEGFR3 − rECs around 12-week-old TB. f , Proliferating rECs (white arrowheads) near 6-week-old TB. Cdh5-mTnG reporter (nGFP, red) shows EC nuclei co-stained with KI-67 (green). g , Scheme of genetic fate-mapping strategy. 4-OHT administration (1 mg per mouse) is indicated by black arrow and red arrows mark time points of analysis. h , i , Tile-scan confocal images ( h ) and higher magnification of insets ( i ) showing fate-tracked Flt4-CreERT2 R26-mTmG (GFP, green)-labelled ECs in femur at 8 weeks (48 h after Cre induction) and 12 weeks (4 weeks after Cre induction). White arrowheads indicate EMCN + CAV1 + rECs and yellow arrowheads mark GFP + traced rECs. j , Quantitative analysis of GFP + rECs (EMCN + CAV1 + FLT4-GFP + ) at 48 h and 4 weeks post-induction, respectively. n = 3 mice per group. Mean ± s.e.m. P values were obtained using an unpaired two-tailed t -test. k , Schematic illustration of genetic fate mapping of rECs in Flt4-CreERT2 R26-mTmG femur.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Injection, Staining, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , b , High-resolution confocal images showing EMCN + VEGFR3 − rECs (arrowheads) around TB in relation to RUNX2 + osteoprogenitors (green) ( a ) and ATP6V1B1/B2 + osteoclasts (green) ( b ). c , d , Maximum-intensity projection showing Sp7-mCherry + osteoblasts (yellow) and ATP6V1B1/B2 + osteoclasts (green) in in relation to EMCN + VEGFR3 − rECs around 6-week-old femoral TB ( c ). Single xy plane ( d ). Green arrowheads mark rECs near osteoclasts and yellow arrowheads near osteoblasts. e , f , Distribution of EMCN + VEGFR3 − type R vessels in relation to Sp7-mCherry + osteoblasts (yellow arrowheads) and ATP6V1B1/B2 + osteoclasts (green arrowheads) around 6-week-old and 12-week-old TB ( e ). Isolated xy plane ( f ). g , Confocal images showing VEGFR3 − VEGFR2 + ECs (white arrowheads) around TB in young and aged patient samples. VEGFR3 (yellow), VEGFR2 (red) and nuclei (DAPI, blue). DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Isolation

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , High-magnification two-photon microscopy images of compact bone immunostained for EMCN (red) and CAV1 (blue) together with second-harmonic generation (SHG, white). Type R capillaries (arrowheads) and trabecular bone (TB) are indicated. Scale bars, 50μm. b, c , Maximum-intensity projections of sections from 3, 6, 8 or 12-week-old femur immunostained for EMCN (red) and OSTERIX (green) ( b ) or EMCN (red), VEGFR3 (blue) and ATP6V1B1/B2 (green) ( c ). Arrowheads mark type R capillaries near trabecular bone (TB). Scale bars, 50μm. Diagram on the right depicts association of rECs with osteoblast-lineage cells and osteoclasts. d . Injection scheme and time points of imaging after labelling of wild-type mice with Calcein Green (CG) and Alizarin Red (AZ). Tile scan imaging of sectioned femur from 8-week-old wild-type after double labelling. Insets depict active resorption in the metaphysis with CG − AR high labelling (top row), active osteogenesis (CG high AR high ) in compact bone (middle row), and active remodelling (CG low AR high ) of trabecular bone (bottom row). n=3 mice. Scale bars, 500μm (overview images) and 50μm (insets).

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Microscopy, Injection, Imaging

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . High-resolution confocal images showing VEGFR3 (yellow) and VEGFR2 (red) immunostaining together with DAPI (blue). VEGFR3 − VEGFR2 + capillaries (white arrowheads) around trabecular bone (TB) and nearby VEGFR3 + VEGFR2 + vessels (yellow arrowheads) in samples from young ( a ) and aged patients ( b ) are indicated. Scale bars, 200μm. c . Tile scan confocal images of EMCN + VEGFR3 − ECs in 12-week-old female and male murine femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. d . Quantitation of EMCN + VEGFR3 − vessel density in 12-week-old female and male femur (n =3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. e . EMCN (red) and VEGFR3 (blue) immunostaining showing the presence of VEGFR3 − EMCN + vessels (white arrowheads) around trabecular bone (TB) in female and male femur. Scale bars, 100μm.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Immunostaining, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP plot (integrated scRNA-seq dataset of all age groups) showing Dach1 expression in rECs (arrow). Colour bar illustrates the expression level. b , Representative confocal images of DACH1 immunostaining (green) in 6-week-old Cdh5-mTnG (red) reporter femur. DACH1 + rECs near TB (green) are marked by white arrowheads. c , Scheme of tamoxifen-induced EC-specific Dach1 inactivation. d , Tile-scan confocal images of EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) and EMCN − VEGFR3 − CAV1 + aECs (yellow arrowheads) in 12-week-old Dach1 iΔEC loss-of-function and littermate control femur. Growth plate (GP) is indicated. e , Quantitation of EMCN + vessel density, EMCN + VEGFR3 − vessel density, and number of CAV1 + arteries in 12-week-old Dach1 iΔEC and control femur ( n = 3–4 female mice per group). Mean ± s.e.m. P values, unpaired two-tailed t -test. Emcn + vessel density plotted with Welch’s correction. f , High-magnification images of metaphysis near GP (left) and of EMCN + VEGFR3 − vessels (white arrowheads; right) around TB in 12-week-old male Dach1 iΔEC and control femurs. White dashed lines in d and f indicate type H area. g , Representative 3D reconstruction of µCT measurements of 12-week-old Dach1 iΔEC and control femoral bone. Dashed yellow lines indicate area analysed. h , Quantitation shows relative bone volume, represented as bone volume/tissue volume (BV/TV) and trabecular thickness (per mm) ( n = 3–4 female mice per group and n = 3 male mice per group). Mean ± s.e.m. P values were plotted using an unpaired two-tailed t -test.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Expressing, Immunostaining, Control, Quantitation Assay, Two Tailed Test

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Schematic representation of tamoxifen-inducible Dach1 overexpression in Aplnr + ECs. b , Tile-scan confocal images showing expansion of CAV1 (green, white arrowheads) immunostained arteries and type R capillaries in the Dach1 gain-of-function ( Dach1 OE ) femur relative to littermate control at postnatal day 30 (P30). c , High-magnification confocal images of EMCN (red) and VEGFR3 (green) immunostained femurs showing the expansion of type R capillaries near TB in Dach1 OE mutants relative to littermate control. White arrowheads indicate type R capillaries. d , Quantitation of arteries/arterioles and CAV1 + area in control and Dach1 OE mutants. n = 3 mice per group. Mean ± s.e.m. P values were obtained by unpaired two-tailed t -test. e , Representative three-dimensional (3D) reconstruction of µCT measurements of P30 Dach1 OE and control femoral TB. f , Quantitation of parameters: bone volume/tissue volume (BV/TV), trabecular number represented by number of trabeculae per millimetre, connectivity density (connectivity density per cubic millimetre) and cortical thickness (mm). n = 3 mice per group. Mean ± s.e.m. P values were obtained by an unpaired two-tailed t -test. g , Schematic overview of scRNA-seq workflow for non-haematopoietic cells from Dach1 OE and littermate control long bone. h , i , UMAP plots of BMSCs ( n = 10,315) with colour-coded subclusters, namely osteoblasts (OBs), osteocytes (OCYs), septoclasts (SCs), proliferating BMSCs (pBMSCs), mpMSCs and dpMSCs (dpMSCs1 and dpMSCs2) ( h ). UMAP distribution of Dach1 OE and control BMSCs, as indicated by colour ( i ). j , Bar plots showing proportion of cells in Dach1 OE and control BMSC subclusters. k , Heatmap showing the top three marker genes for each BMSC subcluster. l , Heatmap illustrating differentially expressed genes in Dach1 OE and control BMSCs related to hypoxia, growth factors, adipogenic and osteogenic transcription factors, and regulators of osteogenesis. Texts in red are the areas of interest. Colour bars in k and l illustrate the expression level (log 2 fold change). Data in h – l are derived from an integrated scRNA-seq dataset of two conditions.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Over Expression, Control, Quantitation Assay, Two Tailed Test, Marker, Expressing, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a, b . Confocal images of femur sections immunostained for EMCN (red) together with OSTERIX (OSX, green) ( a ) or ATP6V1B1/B2 (green) ( b ). Scale bars, 100μm. c . Quantitation of OSX + osteoblast-lineage cells and ATP6V1B1/B2 + osteoclasts in Dach1 OE and control femur (n = 3 in each group). Mean ± SEM. P values, unpaired two-tailed test with Welch’s correction. d-f . Injection scheme and time points of imaging after labelling of Dach1 OE and control with Calcein Green (CG) and Alizarin Red (AZ) ( d ) and tile scan imaging of sectioned femur from P30 Dach1 OE and control after double labelling ( e ). Scale bars, 500μm. Bottom panels in ( e ) show stained trabecular bone. Scale bars, 20μm. f . Quantitation showing extent of bone surface actively mineralizing as mineralizing surface/bone surface (MS/BS) in percentage, mineral apposition rate (MAR) as per micrometre/day, and bone formation rate (BFR) (n=3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test with Welch’s correction for MS/BS, MAR-trabecular bone and BFR. g . Maximum-intensity projections showing prominent HIF1α + immunostaining (yellow arrowheads) in 6-week-old Sp7-mCherry + trabecular osteoblasts, whereas HIF1α + is strongly decreased after emergence of EMCN + VEGFR3 − type R vessels at 12 weeks. Scale bars, 50 μm. h . Maximum-intensity projections of 12-week-old femurs showing immunostaining for hypoxia-related markers in relation to EMCN + VEGFR3 − type R vessels. Shown are immunostaining for Glucose-6-Phosphate Isomerase (GPI), 5′-nucleotidase/CD73 and Haem Oxygenase-1 (HMOX1)-expressing macrophages (yellow arrowheads), all of which are elevated near juvenile trabecular bone relative to the equivalent region in adult. White arrowheads mark EMCN + VEGFR3 − type R vessels. i, j . UMAP plots of bone ECs (n=18383) with colour-coded subclusters ( f ), namely sinusoidal bmECs, metaphyseal mpECs, arterial aECs, remodelling rECs, and proliferating pECs. UMAP distribution of Dach1 OE and control ECs, as indicated by colour ( g ). k . Bar plots showing proportion of cells in Dach1 OE and control samples. l . UMAP plots comparing Dach1 expression in Dach1 OE (bottom) and control (top) EC subclusters. m . Heatmap of differentially expressed genes between EC subclusters. n . Heatmap of differentially expressed genes related to hypoxia and, tissue oxygenation in Dach1 OE and control bone ECs. o . Confocal images showing reduced HIF1α immunostaining (white arrowheads) in Dach1 OE metaphysis and around trabecular bone relative to littermate control. Scale bars, 50μm. Results in panels i, j, m , and n show the integrated mutant and control scRNA-seq data, whereas panels k and l show separated samples derived from the integrated data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Quantitation Assay, Control, Two Tailed Test, Injection, Imaging, Staining, Immunostaining, Expressing, Mutagenesis, Derivative Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a . Violin plots showing selected genes related to reduction of bmEC markers and increased arterialization in Dach1 OE and control. b . Heatmap of differentially expressed genes related to Notch signalling in Dach1 OE and control bone ECs. c . Representative confocal images showing increased Delta-like 4 (DLL4) (green, white arrowheads) expression in vessels around Dach1 OE trabecular bone (TB). Scale bars, 100μm. d . Scheme of tamoxifen-induced (TMX) EC-specific Dll4 inactivation with Cdh5-CreERT2 line. e, f , Tile scan confocal images of EMCN + VEGFR3 − ECs (white arrowheads) (e) and CAV1 + aECs (green arrowheads) (f) in 12-week-old Dll4 iΔEC loss-of-function and control femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. g , Quantitation of EMCN + VEGFR3 − vessel density (left), and number of CAV1 + arteries (right) in 12-week-old Dll4 iΔEC and control femur (n =3 female mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. h . Heatmap showing differentially expressed transcripts for secreted factors in bone EC subpopulations. i, j , Representative images (i) and quantitation (j) of calcified nodules in human mesenchymal stem cells (HMSC) cultured in osteogenic differentiation medium (ODM) supplemented with 100ng/ml Complement C1q and Tumour Necrosis Factor-Related Protein 9 (CTRP9), Neurotrophin 3 (NTF3), Platelet-Derived Growth Factor D (PDGFD), or Semaphorin 7A (SEMA7A) (n=3 independent experiments for each group). Scale bars, 100μm. Graph shows quantitation of absorbance at 405nm. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. k, l , Representative images (k) and quantitation (m) of multinucleated TRAP+ osteoclasts generated from bone marrow-derived monocytes/macrophages treated with RANKL in osteoclast medium (OCM) supplemented with 100ng/ml CTRP9, NTF3, PDGFD, or SEMA7A n=8 (4 independent experiments) for each group. Scale bars, 500μm. Graph shows quantitation of TRAP + multinucleated cells between OCM and treated conditions. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. Panels a and b show separated conditions derived from the integrated mutant and control data, whereas panel h is based on the integrated scRNA-seq data.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Control, Expressing, Quantitation Assay, Two Tailed Test, Cell Culture, Derivative Assay, Generated, Mutagenesis

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , UMAP visualization of bone ECs from 75-week-old bone with colour-coded subclusters. b , Bar plot showing the proportion of cells from each EC subcluster in adult and aged bone. Percentage (%) differences are indicated for rECs and aECs. Data are representative of an individual sample ( a ) and integrated scRNA-seq data from all age groups ( b ). c , d , High-magnification confocal images of TB ( c ) and compact bone (CB) ( d ) immunostained for EMCN (red), VEGFR3 (blue) RUNX2 (yellow arrowheads, marking osteoprogenitors) and ATP6V1B1B2 (green arrowheads, marking osteoclasts). e , High-magnification two-photon microscopy images of CB immunostained for EMCN (red) and VEGFR3 (blue) together with second-harmonic generation (white) showing the changes during ageing. f , Confocal tile-scan images showing EMCN + VEGFR3 − rECs in untreated 12-week-old and 75-week-old mice femur and in response to treatment with alendronate (ALN) or PTH, as indicated. Note expansion of EMCN + VEGFR3 − vessels in CB in response to ageing or treatments (arrowheads). g , Representative confocal images showing EMCN + VEGFR3 − rECs and OSTERIX (green) immunostaining in 75-week-old control, ALN-treated or PTH-treated CB (white arrowheads, marking type R capillaries during ageing). White dashed lines in d – g indicate compact bone (CB) area. h , i , Quantitation of EMCN + CAV1 + vessel density across age groups ( h ), EMCN + VEGFR3 − vessel density in 75-week-old control, ALN-treated or PTH-treated groups, and number of OSTERIX + cells in CB after ALN and PTH treatment compared with respective controls ( i ). n = 3 mice per group. Mean ± s.e.m. P values were obtained using ordinary ANOVA.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Microscopy, Immunostaining, Control, Quantitation Assay

Journal: Nature Cell Biology

Article Title: Specialized post-arterial capillaries facilitate adult bone remodelling

doi: 10.1038/s41556-024-01545-1

Figure Lengend Snippet: a , Scheme for the treatment of adult mice with parathyroid hormone (PTH) or Alendronate. Black arrows indicate onset of treatment, red arrows time points of imaging. b , Confocal images of OSTEOPONTIN (blue) immunostaining of the metaphysis near the growth plate (GP) together with EMCN (red), CAV1 (green) and DAPI (white) to show the response to treatments. Scale bars, 100μm. c , UMAP visualization of sorted bone ECs from control and treatment groups with colour-coded subclusters. d , Bar plots showing the proportions of EC subpopulations in 12-week-old mice. Differences in percentage of rECs and aECs compared to control are indicated. Panels c and d show the individual samples from an integrated scRNA-seq dataset of three conditions. e, f , Confocal images showing EMCN + VEGFR3 − CAV1 + rECs (white arrowheads) around trabecular bone (TB) ( e ) and association of ATP6V1B1/B2 + (green) osteoclasts with EMCN + VEGFR3 − type R capillaries ( f ) in 12-week-old control and indicated treatment conditions. Scale bars, 50μm. g , Effect of treatments on EMCN + VEGFR3 − rECs (white arrowheads) and RUNX2 + (green) osteoprogenitors around trabecular bone (TB). Scale bars, 100μm.

Article Snippet: Sections were incubated in blocking buffer (PBS, 0.2% CHAPS, 5% donkey serum, 10% DMSO and 25 mM EDTA, pH 8) for 1 h before staining with primary antibodies mouse anti-VEGFR3 (R&D Systems, MAB3491, 1:100 dilution) and goat anti-VEGFR2 (R&D Systems, AF357; 1:100 dilution) overnight.

Techniques: Imaging, Immunostaining, Control

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Volcano plot of proteomic analysis of murine plasma from WT and B2KO mice ( n = 6). VEGFR3 (FLT4) is highlighted in red. ( B ) Normalized VEGFR3 LFQ intensities extracted from A . ( C ) MSD-assay quantifications of sVEGFR3 in the same plasma samples. ( D ) Immunoblot detection of sVEGFR3 ectodomain in mouse plasma from A , using nonreducing and reducing conditions. ( E ) Volcano plot of proteomic analysis of murine plasma from an independent B2KO line ( n = 9) compared with WT ( n =9) and ( F ) the extracted normalized LFQ values. Volcano plots of the proteomic analyses of Bace1/Bace2 double-knockout (BDKO) mice ( n =9) compared with the WT line ( n = 9) ( G ) (corresponding extracted LFQ intensities of sVEGFR3 in F ) and B1KO ( n = 9) compared with an individual control WT line ( n = 9) ( H ). ( I ) Normalized LFQ values extracted from H . ( J ) Localization of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The signal peptide is shown in rose, the ectodomain is indicated in blue, the intracellular domain in green, and the transmembrane domain in yellow. Two sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots ( A , E , G , and H ). Proteins with P < 0.05 are shown as red circles. Extracted LFQ quantifications ( B , F , and I ) of VEGFR3 with significance after FDR correction are labeled with plus signs. All dot plots were normalized on the WT mean and depict mean and SD. MSD-assay data ( C ) additionally depicts the P value calculated by unpaired t test. **** P < 0.0001.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Western Blot, Double Knockout, Control, Sequencing, Labeling

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Schematic of VEGFR3 fragments. From left to right: The immature proVEGFR3 (200 kDa) can be cleaved by BACE2, releasing the immature, soluble ectodomain sol proVEGFR3 (130 kDa). The mature protein consists of 2 subunits linked through a disulfide bridge: VEGFR3α (75 kDa) and VEGFR3β (125 kDa). Upon BACE2 cleavage, the VEGFR3β-CTF (70 kDa) and sVEGFR3 (130 kDa) are generated, the latter of which consists of the VEGFR3α (75 kDa) and VEGFR3β-NTF (55 kDa) fragments. ( B ) Immunoblot detection of VEGFR3 in lysates and media of HEK293 cells transfected with empty control plasmids (Ctrl), Vegfr3 , Vegfr3 + Bace2 , and Bace2 . B2, BACE2. Data show 3 independent experiments. sVEGFR3 is not detectable under reducing conditions. sol proVEGFR3 in the lysates appears at around 100 kDa and derives from BACE2 cleavage of immaturely glycosylated proVEGFR3 early in the secretory pathway upon BACE2 overexpression. ( C ) Localization and length of identified individual peptides (black dots) on the canonical VEGFR3 sequence. The ectodomain is indicated in blue, the intracellular domain in green, the signal peptide in orange, and the transmembrane domain in yellow. ( D ) N-terminal juxtamembrane region of VEGFR3 sequence. The identified semispecific peptide after LysN digestion is marked in yellow, the proposed cleavage site after amino acid alanine with 2 vertical lines, and the transmembrane region in gray. ( E ) Comparison of the fragment ion spectra of the identified C-terminal peptide of the LysN digestion KGC(cam)VN(+1)SSASVA (lower spectrum) to a synthetic peptide with the same sequence (upper spectrum). Identified y-ions are indicated in red, b-ions in blue, and fragment ions with neutral losses in green. Both spectra match with a dot product of 0.93 for the fragment ion intensities.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Generated, Western Blot, Transfection, Control, Over Expression, Sequencing, Comparison

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Immunoblot detection after control treatment (–) or upon BACE1 and BACE2 knockdown (+). Lysates were blotted for VEGFR3, BACE1/2, and actin. Conditioned media were blotted for sVEGFR3. ( B ) Corresponding densitometric quantifications, deriving from VEGFR3β (lysate) and sVEGFR3 (medium). ( C ) Immunoblots of cells treated with DMSO (–) or 100 nM verubecestat (+). ( D ) Corresponding densitometric quantifications as in B . Dot plots were normalized on the control mean and depict mean and SD, alongside the calculated P values, calculated by unpaired t test. ** P < 0.01; **** P < 0.0001. P values are only indicated where significance was observed. Data are derived from n = 6 biological replicates. Shown are representative data from 3 independent experiments.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Western Blot, Control, Knockdown, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: ( A ) Schematic for VEGFR3 signaling. Upon ligand binding, VEGFR3 dimerizes, resulting in intracellular autophosphorylation and activation of the downstream genes FOXC2 and DLL4 . V, verubecestat, inhibitor of BACE2. Gene expression levels of ( B ) DLL4 and FOXC2 and ( C ) VEGFR3 after the application of DMSO, 100 nM verubecestat (V), and VEGF-C. ( D and E ) Gene expression levels of DLL4 , FOXC2 , VEGFR3 , BACE1 , and BACE2 after BACE knockdown (siB1/siB2) without or with (+V) subsequent verubecestat application. All dot plots were normalized on the control mean and depict mean and SD alongside the P values calculated by unpaired t tests against the DMSO control ( B and C ) or by 1-way ANOVA ( D and E ), in both cases followed by Bonferroni’s multiple-comparison test. * P < 0.05; ** P < 0.01; **** P < 0.0001. P values are only indicated where significance was observed. In B , 1 data point was excluded from the DLL4 expression/VEGF-C156S data set, since it was identified as an outlier via the ROUT method. Data are derived from n = 6 ( B and C ) or n = 4 ( D and E ) biological replicates.

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Ligand Binding Assay, Activation Assay, Expressing, Knockdown, Control, Comparison, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: The Alzheimer’s disease–linked protease BACE2 cleaves VEGFR3 and modulates its signaling

doi: 10.1172/JCI170550

Figure Lengend Snippet: Volcano plots of proteomic analysis of murine plasma from ( A ) compound 89–treated (Cpd89) versus vehicle-treated (veh) mice and ( B ) LY2811376-treated versus vehicle-treated mice ( n = 13, treated; n = 14, veh). VEGFR3 is highlighted in red. ( C ) Corresponding extracted LFQ intensities of sVEGFR3 and ( D ) MSD-assay quantifications of sVEGFR3. Plasma sVEGFR3 ( E ) and plasma sSEZ6L ( F ) levels in 8–10 B1KO, B2KO, and respective WT mice with (blue) or without (black) 3 days of 50 mg/kg per os twice a day verubecestat dosing. ( G ) Plasma levels of VEGFR3 and SEZ6L during 7 days of 0.1% dietary verubecestat (average drug intake, 97 mg/kg/d; n = 6 per group, all male, age: 7–10 weeks), respective to untreated control levels. Two-sided Student’s t tests with a permutation-based FDR correction (FDR < 0.05; indicated by hyperbolic curves) were used for volcano plots ( A and B ). Proteins with P < 0.05 are shown as red circles. ( C ) Significance after FDR correction is indicated with plus signs. All dot plots were normalized on the control mean and depict the SD alongside the calculated P values, calculated by 1-way ( D and G ) or 2-way ( E and F ) ANOVA with Bonferroni’s multiple-comparison test. * P < 0.05; *** P < 0.001; **** P < 0.0001. P values are only indicated where significance could be observed. Number of biological replicates in E and F was 9, except for Bace1-WT + verubecestat ( n = 8) and for Bace2WT without verubecestat ( n = 10).

Article Snippet: For human VEGFR3 MSD-assays VEGFR3 was detected using goat polyclonal anti-human VEGFR3/Flt-4 biotinylated antibody (BAF349, R&D Systems).

Techniques: Control, Comparison