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PreprintCited by 134Lymphatic System and DiseasesLymphatic Disorders and TreatmentsVascular Malformations and Hemangiomas

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Angiopoietin 2 regulates the transformation and integrity of lymphatic endothelial cell junctions

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Zheng, Wei, Nurmi, Harri, Appak, Sila, Sabine, Amélie, Bovay, Esther, Korhonen, Emilia A., Orsenigo, Fabrizio, Lohela, Marja, D’Amico, Gabriela, Holopainen, Tanja, Leow, Ching Ching, Dejana, Elisabetta, Petrova, Tatiana V., Augustin, Hellmut G., Alitalo, Kari·2014·Genes & Development
DOI 10.1101/gad.237677.114PMC PMC4102766
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Abstract

Primitive lymphatic vessels are remodeled into functionally specialized initial and collecting lymphatics during development. Lymphatic endothelial cell (LEC) junctions in initial lymphatics transform from a zipper-like to a button-like pattern during collecting vessel development, but what regulates this process is largely unknown. Angiopoietin 2 ( Ang2 ) deficiency leads to abnormal lymphatic vessels. Here we found that an ANG2-blocking antibody inhibited embryonic lymphangiogenesis, whereas endothelium-specific ANG2 overexpression induced lymphatic hyperplasia. ANG2 inhibition blocked VE-cadherin phosphorylation at tyrosine residue 685 and the concomitant formation of button-like junctions in initial lymphatics. The defective junctions were associated with impaired lymph uptake. In collecting lymphatics, adherens junctions were disrupted, and the vessels leaked upon ANG2 blockade or gene deletion. ANG2 inhibition also suppressed the onset of lymphatic valve formation and subsequent valve maturation. These data identify ANG2 as the first essential regulator of the functionally important interendothelial cell–cell junctions that form during lymphatic development.

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Figures

Figure 1. ANG2 regulates lymphangiogenesis during embryonic development. (A–H) Pregnant females were injected intraperitoneally (i.p.) with 30 mg/kg ANG2-blocking antibody or a control antibody at E12.5, E14.5, and E16.5, and the ventral skin of embryos was analyzed at E18.5 by immunofluorescence for LYVE1, VEGFR3, Ki67, and PROX1, as indicated. Dashed lines indicate the umbilicus. Vessels inside the red squares are quantified in I. (Yellow arrows) Sprouts; (white arrows) Ki67+ LECs; (arrowheads) Ki67− LECs. Bars: A,E, 200 µm; B,C,F,G, 20 µm; D,H, 10 µm. (I) Quantification of the migration, branch points, and sprouts; the number of LECs per 1-mm vessel; and the percentage of Ki67+ LECs out of all LECs. N = 4–5. (J–L) The mothers of VE-cadherin-tTA; TetO-Ang2 embryos were provided with tetracycline water until E10.5, and the ventral skin of the embryos was analyzed at E18.5. The anti-VEGFR3 antibody (30 mg/kg) was injected i.p. at E14.5 and E16.5. (DTG) Double transgenic for VE-cadherin-tTA and TetO-Ang2; (Ctrl) control embryos (wild type [WT] or single transgenic). N = 4–7. Boxed regions are magnified in the bottom panel. Bars: top panel, 200 µm; bottom panel, 20 µm. All values are mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.
Figure 1. ANG2 regulates lymphangiogenesis during embryonic development. (A–H) Pregnant females were injected intraperitoneally (i.p.) with 30 mg/kg ANG2-blocking antibody or a control antibody at E12.5, E14.5, and E16.5, and the ventral skin of embryos was analyzed at E18.5 by immunofluorescence for LYVE1, VEGFR3, Ki67, and PROX1, as indicated. Dashed lines indicate the umbilicus. Vessels inside the red squares are quantified in I. (Yellow arrows) Sprouts; (white arrows) Ki67+ LECs; (arrowheads) Ki67− LECs. Bars: A,E, 200 µm; B,C,F,G, 20 µm; D,H, 10 µm. (I) Quantification of the migration, branch points, and sprouts; the number of LECs per 1-mm vessel; and the percentage of Ki67+ LECs out of all LECs. N = 4–5. (J–L) The mothers of VE-cadherin-tTA; TetO-Ang2 embryos were provided with tetracycline water until E10.5, and the ventral skin of the embryos was analyzed at E18.5. The anti-VEGFR3 antibody (30 mg/kg) was injected i.p. at E14.5 and E16.5. (DTG) Double transgenic for VE-cadherin-tTA and TetO-Ang2; (Ctrl) control embryos (wild type [WT] or single transgenic). N = 4–7. Boxed regions are magnified in the bottom panel. Bars: top panel, 200 µm; bottom panel, 20 µm. All values are mean ± SEM. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.CC BY-NC · PubMed Central
Figure 2. ANG2-inhibited embryos fail to form button-like junctions in initial lymphatics. (A–H) Pregnant females were treated with the ANG2-blocking or a control antibody at E12.5, E14.5, and E16.5, and the ventral skin of embryos was analyzed at E18.5. N = 15–16 from five independent experiments. (I–P) Females were treated with the indicated antibodies as above together with 1 mg/kg dexamethasone. N = 4. Boxed regions in A–G and I–O are magnified in the panels below them. (Arrows) Zippers; (arrowheads) buttons.
Figure 2. ANG2-inhibited embryos fail to form button-like junctions in initial lymphatics. (A–H) Pregnant females were treated with the ANG2-blocking or a control antibody at E12.5, E14.5, and E16.5, and the ventral skin of embryos was analyzed at E18.5. N = 15–16 from five independent experiments. (I–P) Females were treated with the indicated antibodies as above together with 1 mg/kg dexamethasone. N = 4. Boxed regions in A–G and I–O are magnified in the panels below them. (Arrows) Zippers; (arrowheads) buttons.CC BY-NC · PubMed Central
Figure 3. Ang2 gene deletion impairs the zipper-to-button junctional transformation in initial lymphatics. (A) The diaphragms of Ang2−/− or wild-type (WT) P5 pups were whole-mount-stained. (Arrows) Sprouting front. Boxed regions are magnified in the left panel. N = 4–5. (B–K) The ear skin of 8-wk-old Ang2−/− mice (B–G) and their wild-type littermates (H–J) was whole-mount-stained. (Arrows) Zippers; (arrowheads) transforming junctions; (empty arrowheads) buttons. (K) Quantification of 36–43 initial lymphatics from four mice of each genotype, analyzed for their junctional patterns. (***) P < 0.001. Bars, 20 µm in all.
Figure 3. Ang2 gene deletion impairs the zipper-to-button junctional transformation in initial lymphatics. (A) The diaphragms of Ang2−/− or wild-type (WT) P5 pups were whole-mount-stained. (Arrows) Sprouting front. Boxed regions are magnified in the left panel. N = 4–5. (B–K) The ear skin of 8-wk-old Ang2−/− mice (B–G) and their wild-type littermates (H–J) was whole-mount-stained. (Arrows) Zippers; (arrowheads) transforming junctions; (empty arrowheads) buttons. (K) Quantification of 36–43 initial lymphatics from four mice of each genotype, analyzed for their junctional patterns. (***) P < 0.001. Bars, 20 µm in all.CC BY-NC · PubMed Central
Figure 4. Defective lymph uptake in anti-ANG2-treated embryos. Pregnant females were treated with IgG (A–C) or the ANG2-blocking antibody (D–F) at E12.5, E14.5, and E17.5. At E19.5, the embryos were injected with FITC-Dextran in the periorbital dermis (arrows in A,D) or forelimb footpad (arrowheads in A,D) and imaged to check the tracer uptake by lymphatic vessels. (B,C) Arrows indicate the tracer drained by the lymphatic vessels in the IgG-treated embryos (12 of 13). (E,F) No tracer uptake was observed in the anti-ANG2-treated embryos (N = 11). (*) Injection sites. Bars: A,D, 2 mm; B,C,E,F, 1 mm.
Figure 4. Defective lymph uptake in anti-ANG2-treated embryos. Pregnant females were treated with IgG (A–C) or the ANG2-blocking antibody (D–F) at E12.5, E14.5, and E17.5. At E19.5, the embryos were injected with FITC-Dextran in the periorbital dermis (arrows in A,D) or forelimb footpad (arrowheads in A,D) and imaged to check the tracer uptake by lymphatic vessels. (B,C) Arrows indicate the tracer drained by the lymphatic vessels in the IgG-treated embryos (12 of 13). (E,F) No tracer uptake was observed in the anti-ANG2-treated embryos (N = 11). (*) Injection sites. Bars: A,D, 2 mm; B,C,E,F, 1 mm.CC BY-NC · PubMed Central
Figure 5. ANG2 is required for VE-cadherin Y685 phosphorylation in developing initial lymphatics. Pregnant females were treated with the control antibody (A–C,G–I) or the ANG2-blocking antibody (D–F,J–L) at E12.5, E14.5, and E16.5, and the ventral skin from the embryos was analyzed at E18.5. Bars, 20 µm. (M) Quantification of the intensities of VE-cadherin P-Y685-positive pixels out of those of the total VE-cadherin-positive pixels. N = 6, pooled from two independent experiments with consistent results. (***) P < 0.001.
Figure 5. ANG2 is required for VE-cadherin Y685 phosphorylation in developing initial lymphatics. Pregnant females were treated with the control antibody (A–C,G–I) or the ANG2-blocking antibody (D–F,J–L) at E12.5, E14.5, and E16.5, and the ventral skin from the embryos was analyzed at E18.5. Bars, 20 µm. (M) Quantification of the intensities of VE-cadherin P-Y685-positive pixels out of those of the total VE-cadherin-positive pixels. N = 6, pooled from two independent experiments with consistent results. (***) P < 0.001.CC BY-NC · PubMed Central
Figure 6. Disruption of adherens junctions in the collecting lymphatics of anti-ANG2-treated and Ang2−/− mice. (A–D) Neonatal mice were injected with IgG or the anti-ANG2 antibody every other day starting from P0 and were analyzed at P5. (Arrows) Leak of chyle; (arrowheads) normal drainage of chyle by the collecting lymphatics. (E–H) Whole-mount staining of the mesenteries in A and B for VEGFR3, VE-cadherin, and PROX1. (Arrowheads) Disrupted interendothelial junctions; (arrows) valves. Boxed regions are magnified in E and F. N = 7–9, two independent experiments. (I) Whole-mount staining of the mesentery of Ang2−/− or wild-type (WT) pups at P5. (Arrowheads) Disrupted junctions. (J–O) Collecting lymphatics in the ear skin of 8-wk-old Ang2−/− and wild-type littermate mice. (Arrows) Zippers; (arrowheads) disrupted junctions; (empty arrowheads) button junctions. N = 4–5. (P) FITC-Dextran lymphangiography of wild-type and Ang2−/− ears. Note the leaked tracer, as indicated by asterisks. N = 6. (Q) Evans Blue content in the footpad and in blood circulation after intradermal injection in the lower limbs. N = 6. (***) P < 0.001. Bars: A,B, 2 mm; C,D, 0.5 mm; E,F,I–O, 20 µm; G,H,P, 100 µm.
Figure 6. Disruption of adherens junctions in the collecting lymphatics of anti-ANG2-treated and Ang2−/− mice. (A–D) Neonatal mice were injected with IgG or the anti-ANG2 antibody every other day starting from P0 and were analyzed at P5. (Arrows) Leak of chyle; (arrowheads) normal drainage of chyle by the collecting lymphatics. (E–H) Whole-mount staining of the mesenteries in A and B for VEGFR3, VE-cadherin, and PROX1. (Arrowheads) Disrupted interendothelial junctions; (arrows) valves. Boxed regions are magnified in E and F. N = 7–9, two independent experiments. (I) Whole-mount staining of the mesentery of Ang2−/− or wild-type (WT) pups at P5. (Arrowheads) Disrupted junctions. (J–O) Collecting lymphatics in the ear skin of 8-wk-old Ang2−/− and wild-type littermate mice. (Arrows) Zippers; (arrowheads) disrupted junctions; (empty arrowheads) button junctions. N = 4–5. (P) FITC-Dextran lymphangiography of wild-type and Ang2−/− ears. Note the leaked tracer, as indicated by asterisks. N = 6. (Q) Evans Blue content in the footpad and in blood circulation after intradermal injection in the lower limbs. N = 6. (***) P < 0.001. Bars: A,B, 2 mm; C,D, 0.5 mm; E,F,I–O, 20 µm; G,H,P, 100 µm.CC BY-NC · PubMed Central
Figure 7. ANG2 regulates various steps of valve morphogenesis. (A–F) Pregnant mice were treated with the indicated antibodies every other day starting at E12.5, and the mesenteries were analyzed at E18.5. N = 3–4. (Arrows) Valves; (arrowheads) FOXC2+ ECs in the arteries. Lymphatic vessels are outlined by dashed lines (a). Boxed regions are magnified in the corresponding insets. Bars, 20 µm. (G) Quantification of A–F. Mean ± SEM. (**) P < 0.01. (H) Pregnant mice were treated with the indicated antibodies from E14.5 to E18.5. Bars: top panel, 100 µm; bottom panel, 20 µm. Boxed regions are magnified below. (Arrow) Valve. Bars: top panel, 100 µm; bottom panel, 20 µm. (I–P) Pregnant mice were treated with the indicated antibodies from E15.5 to E18.5. (Arrowheads) Clusters of lymphatic valve-forming cells that failed to reorientate, express integrin-α9, and form valve leaflets; (open arrowheads) “ring-shaped” valves that expressed integrin-α9 but did not elongate leaflets; (arrows) mature valves with elongated leaflets. (Q) Evaluation and quantification of valve maturation in I–P. Thirty-two to 44 valves were analyzed from three to four embryos of each antibody treatment. (***) P < 0.001.
Figure 7. ANG2 regulates various steps of valve morphogenesis. (A–F) Pregnant mice were treated with the indicated antibodies every other day starting at E12.5, and the mesenteries were analyzed at E18.5. N = 3–4. (Arrows) Valves; (arrowheads) FOXC2+ ECs in the arteries. Lymphatic vessels are outlined by dashed lines (a). Boxed regions are magnified in the corresponding insets. Bars, 20 µm. (G) Quantification of A–F. Mean ± SEM. (**) P < 0.01. (H) Pregnant mice were treated with the indicated antibodies from E14.5 to E18.5. Bars: top panel, 100 µm; bottom panel, 20 µm. Boxed regions are magnified below. (Arrow) Valve. Bars: top panel, 100 µm; bottom panel, 20 µm. (I–P) Pregnant mice were treated with the indicated antibodies from E15.5 to E18.5. (Arrowheads) Clusters of lymphatic valve-forming cells that failed to reorientate, express integrin-α9, and form valve leaflets; (open arrowheads) “ring-shaped” valves that expressed integrin-α9 but did not elongate leaflets; (arrows) mature valves with elongated leaflets. (Q) Evaluation and quantification of valve maturation in I–P. Thirty-two to 44 valves were analyzed from three to four embryos of each antibody treatment. (***) P < 0.001.CC BY-NC · PubMed Central

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Authors
Zheng, W., Nurmi, H., Appak, S., Sabine, A., Bovay, E., Korhonen, E. A., Orsenigo, F., Lohela, M., D’Amico, G., Holopainen, T., Leow, C. C., Dejana, E., Petrova, T. V., Augustin, H. G., & Alitalo, K.
Affiliations
  • University of Helsinki (FI)
  • Centre Hospitalier Universitaire Vaudois (CH)
  • University of Milan (IT)
  • German Cancer Research Center (DE)
Corresponding author
Alitalo, Kari
ORCID
Venue
Genes & Development
Publisher
Cold Spring Harbor Laboratory
Published
2014-07-15
Volume
28
Issue
14
Pages
1592-1603
ISSN
  • 0890-9369 (ISSN-L)
  • 1549-5477
DOI
10.1101/gad.237677.114
PMC
PMC4102766
OpenAlex
W2066164950
Cited by
134
References
46
Citations by year
2026: 9 · 2025: 9 · 2024: 7 · 2023: 16 · 2022: 13 · 2021: 12 · 2020: 15 · 2019: 12 · 2018: 12 · 2017: 10 · 2016: 9 · 2015: 9 · 2014: 1
Type
posted content
Language
en
Open access
Licence
cc-by-nc
Locations
  • Genes & Development (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
  • SERVAL (Université de Lausanne) (publishedVersion, cc-by-nc)
  • IRIS (submittedVersion, cc-by-nc)
  • Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
  • HighWire Press Open Archive (submittedVersion)
Funders
  • European Commission
  • Fondation Leducq (11CVD03)
  • Academy of Finland
  • Biomedicum Helsinki-säätiö
  • Orionin Tutkimussäätiö
  • FP7 Ideas: European Research Council (268804)
Topics
  • Health Sciences › Medicine › Oncology › Lymphatic System and Diseases
  • Health Sciences › Medicine › Surgery › Lymphatic Disorders and Treatments
  • Health Sciences › Medicine › Surgery › Vascular Malformations and Hemangiomas
Keywords
angiopoietin-2, lymphatic endothelial cells, lymphatic development, lymphangiogenesis, lymphoid hyperplasia, VE-cadherin phosphorylation, adherens junctions, lymphatic leakage, lymphatic absorption
MeSH terms
Animals, Embryo, Mammalian, Intercellular Junctions, Lymphoid Tissue, Mice, Inbred C57BL, Phosphorylation, Cadherins, Gene Deletion, Lymphangiogenesis, Angiopoietin-2, Endothelial Cells, Mice

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