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Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

Xu Y, Yuan L, Mak J, Pardanaud L, Caunt M, Kasman I, Larrivée B, Del Toro R, Suchting S, Medvinsky A, Silva J, Yang J, Thomas JL, Koch AW, Alitalo K, Eichmann A, Bagri A - J. Cell Biol. (2010)

Bottom Line: Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment.In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs.Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 833, 75005 Paris, France.

ABSTRACT
Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

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Normal lymphatic development in double-heterozygous nrp2+/−vegfr2+/− mice. (A) EGFP staining of mesenteric vessels in a heterozygous vegfr2-egfp mouse pup at P0. Note green fluorescence in capillaries covering the surface of the duodenum and in mesenteric arteries, veins, and lymphatic vessels. D, duodenum; A, mesenteric arteries; V, veins; L, lymphatic vessels. (B–B″) EGFP–LYVE-1 double staining of tail skin at P1. Note Flk-1–EGFP expression in a lymphatic vessel sprout (asterisks) and in filopodia-extending tips (arrows). (C) Quantification of lymph vessel branch points. Error bars indicate SEM. (D–D″) Normal appearance of lymphatic vessel sprouts (asterisks) and filopodia-extending tips (arrows) in P1 tail skin in double-heterozygous nrp2+/−vegfr2+/− mice. (E and F) Lower magnification views of LYVE-1–positive lymphatic vessels in wild-type (E) and nrp2+/−vegfr2+/− dorsal trunk skin. WT, wild type. (F’–F″) Flk-1/VEGFR2-EGFP–LYVE-1 double labeling. OV, overlay. Bars: (A) 200 µm; (B–D″) 50 µm; (E–F″) 200 µm.
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fig5: Normal lymphatic development in double-heterozygous nrp2+/−vegfr2+/− mice. (A) EGFP staining of mesenteric vessels in a heterozygous vegfr2-egfp mouse pup at P0. Note green fluorescence in capillaries covering the surface of the duodenum and in mesenteric arteries, veins, and lymphatic vessels. D, duodenum; A, mesenteric arteries; V, veins; L, lymphatic vessels. (B–B″) EGFP–LYVE-1 double staining of tail skin at P1. Note Flk-1–EGFP expression in a lymphatic vessel sprout (asterisks) and in filopodia-extending tips (arrows). (C) Quantification of lymph vessel branch points. Error bars indicate SEM. (D–D″) Normal appearance of lymphatic vessel sprouts (asterisks) and filopodia-extending tips (arrows) in P1 tail skin in double-heterozygous nrp2+/−vegfr2+/− mice. (E and F) Lower magnification views of LYVE-1–positive lymphatic vessels in wild-type (E) and nrp2+/−vegfr2+/− dorsal trunk skin. WT, wild type. (F’–F″) Flk-1/VEGFR2-EGFP–LYVE-1 double labeling. OV, overlay. Bars: (A) 200 µm; (B–D″) 50 µm; (E–F″) 200 µm.

Mentions: We intercrossed heterozygous nrp2+/− mice with vegfr2+/egfp mice to test for possible defects in lymphatic vessel development in double-heterozygous offspring. Whole-mount analysis of P1 guts showed egfp expression in mesenteric arteries, veins, and capillaries covering the gut surface as well as robust egfp expression in lymphatic mesenteric vessels (Fig. 5 A). Similarly, double labeling of skin isolated from the trunk or tail of vegfr2+/egfp mice with the lymphatic marker LYVE-1 showed double labeling of lymphatic vessels with both egfp and LYVE-1, extending into the tips of sprouting lymphatic vessels (Fig. 5, B–B″). Arteries, veins, and capillaries expressed only egfp. Thus, consistent with the results obtained using antibody labeling (Fig. S2), vegfr2 expression was routinely detected by this reporter construct in lymphatic vessels including sprouting tips.


Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.

Xu Y, Yuan L, Mak J, Pardanaud L, Caunt M, Kasman I, Larrivée B, Del Toro R, Suchting S, Medvinsky A, Silva J, Yang J, Thomas JL, Koch AW, Alitalo K, Eichmann A, Bagri A - J. Cell Biol. (2010)

Normal lymphatic development in double-heterozygous nrp2+/−vegfr2+/− mice. (A) EGFP staining of mesenteric vessels in a heterozygous vegfr2-egfp mouse pup at P0. Note green fluorescence in capillaries covering the surface of the duodenum and in mesenteric arteries, veins, and lymphatic vessels. D, duodenum; A, mesenteric arteries; V, veins; L, lymphatic vessels. (B–B″) EGFP–LYVE-1 double staining of tail skin at P1. Note Flk-1–EGFP expression in a lymphatic vessel sprout (asterisks) and in filopodia-extending tips (arrows). (C) Quantification of lymph vessel branch points. Error bars indicate SEM. (D–D″) Normal appearance of lymphatic vessel sprouts (asterisks) and filopodia-extending tips (arrows) in P1 tail skin in double-heterozygous nrp2+/−vegfr2+/− mice. (E and F) Lower magnification views of LYVE-1–positive lymphatic vessels in wild-type (E) and nrp2+/−vegfr2+/− dorsal trunk skin. WT, wild type. (F’–F″) Flk-1/VEGFR2-EGFP–LYVE-1 double labeling. OV, overlay. Bars: (A) 200 µm; (B–D″) 50 µm; (E–F″) 200 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812843&req=5

fig5: Normal lymphatic development in double-heterozygous nrp2+/−vegfr2+/− mice. (A) EGFP staining of mesenteric vessels in a heterozygous vegfr2-egfp mouse pup at P0. Note green fluorescence in capillaries covering the surface of the duodenum and in mesenteric arteries, veins, and lymphatic vessels. D, duodenum; A, mesenteric arteries; V, veins; L, lymphatic vessels. (B–B″) EGFP–LYVE-1 double staining of tail skin at P1. Note Flk-1–EGFP expression in a lymphatic vessel sprout (asterisks) and in filopodia-extending tips (arrows). (C) Quantification of lymph vessel branch points. Error bars indicate SEM. (D–D″) Normal appearance of lymphatic vessel sprouts (asterisks) and filopodia-extending tips (arrows) in P1 tail skin in double-heterozygous nrp2+/−vegfr2+/− mice. (E and F) Lower magnification views of LYVE-1–positive lymphatic vessels in wild-type (E) and nrp2+/−vegfr2+/− dorsal trunk skin. WT, wild type. (F’–F″) Flk-1/VEGFR2-EGFP–LYVE-1 double labeling. OV, overlay. Bars: (A) 200 µm; (B–D″) 50 µm; (E–F″) 200 µm.
Mentions: We intercrossed heterozygous nrp2+/− mice with vegfr2+/egfp mice to test for possible defects in lymphatic vessel development in double-heterozygous offspring. Whole-mount analysis of P1 guts showed egfp expression in mesenteric arteries, veins, and capillaries covering the gut surface as well as robust egfp expression in lymphatic mesenteric vessels (Fig. 5 A). Similarly, double labeling of skin isolated from the trunk or tail of vegfr2+/egfp mice with the lymphatic marker LYVE-1 showed double labeling of lymphatic vessels with both egfp and LYVE-1, extending into the tips of sprouting lymphatic vessels (Fig. 5, B–B″). Arteries, veins, and capillaries expressed only egfp. Thus, consistent with the results obtained using antibody labeling (Fig. S2), vegfr2 expression was routinely detected by this reporter construct in lymphatic vessels including sprouting tips.

Bottom Line: Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment.In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs.Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 833, 75005 Paris, France.

ABSTRACT
Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.

Show MeSH
Related in: MedlinePlus