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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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Anti-Nrp2B treatment in vitro results in reduced sprout formation and altered tip cell behavior. (A–C) Representative examples of LECs sprouting from coated beads in control (A), VEGF-C (B)–, or anti-Nrp2B (C)–treated cultures stained with anti–LYVE-1. (D) Sprout number is significantly reduced with anti-Nrp2B or VEGFR3 ECD treatment compared with VEGF-C treatment alone. (E) Sprout length is not reduced with anti-Nrp2B treatment but is reduced by VEGFR3 ECD treatment compared with VEGF-C treatment alone (n = 5 beads/well for 10 wells). (F–I) Quantification of sprout initiation events (F), sprout-stalling events (G), and sprout extension rates from live imaging experiments. Error bars indicate SEM. *, P < 0.01. Bar, 150 µm.
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fig4: Anti-Nrp2B treatment in vitro results in reduced sprout formation and altered tip cell behavior. (A–C) Representative examples of LECs sprouting from coated beads in control (A), VEGF-C (B)–, or anti-Nrp2B (C)–treated cultures stained with anti–LYVE-1. (D) Sprout number is significantly reduced with anti-Nrp2B or VEGFR3 ECD treatment compared with VEGF-C treatment alone. (E) Sprout length is not reduced with anti-Nrp2B treatment but is reduced by VEGFR3 ECD treatment compared with VEGF-C treatment alone (n = 5 beads/well for 10 wells). (F–I) Quantification of sprout initiation events (F), sprout-stalling events (G), and sprout extension rates from live imaging experiments. Error bars indicate SEM. *, P < 0.01. Bar, 150 µm.

Mentions: To directly evaluate the cellular basis for why Nrp2 inhibition affects sprout development, we tested the effects of anti-Nrp2B on LEC sprouting using an in vitro sprouting assay. Over the course of 14 d, the LECs form sprouts that radially extend from the bead and often display lumen formation and branching, which is highly reminiscent of lymphatic vessel sprouting in vivo. In the presence of VEGF-C, the LECs proliferate and form robust sprouts that extend from the bead (Fig. 4, A and B). Treatment with anti-Nrp2B reduced the number of sprouts (Fig. 4, C and D). Stimulation by VEGF-C resulted in an increase of sprout number from a mean of 2.4 sprouts per bead to a mean of 6.3 sprouts per bead (P < 0.01). Inhibition of Nrp2 by anti-Nrp2B in the presence of VEGF-C resulted in a significant reduction in the number of sprouts to 3.7 (P < 0.0001 compared with VEGF-C). As a positive control for blocking VEGF-C activity, the VEGFR3 extracellular domain protein (ECD; Mäkinen et al., 2001; Caunt et al., 2008) reduced sprouting to 1.7 sprouts per bead, which is a level comparable with the no VEGF-C control. Nrp2 inhibition did not affect mean sprout length (364 pixels in VEGF-C treated vs. 314 pixels in anti-Nrp2B treated; P = 0.09) in contrast to VEGFR3 ECD (53 pixels; P < 0.0001; Fig. 4 E). These data corroborate our in vivo observations that Nrp2 inhibition results in reduced sprouting but not sprout length, likely not affecting stalk cells. It also suggested an effect on the initial aspects of sprout formation.


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)

Anti-Nrp2B treatment in vitro results in reduced sprout formation and altered tip cell behavior. (A–C) Representative examples of LECs sprouting from coated beads in control (A), VEGF-C (B)–, or anti-Nrp2B (C)–treated cultures stained with anti–LYVE-1. (D) Sprout number is significantly reduced with anti-Nrp2B or VEGFR3 ECD treatment compared with VEGF-C treatment alone. (E) Sprout length is not reduced with anti-Nrp2B treatment but is reduced by VEGFR3 ECD treatment compared with VEGF-C treatment alone (n = 5 beads/well for 10 wells). (F–I) Quantification of sprout initiation events (F), sprout-stalling events (G), and sprout extension rates from live imaging experiments. Error bars indicate SEM. *, P < 0.01. Bar, 150 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Anti-Nrp2B treatment in vitro results in reduced sprout formation and altered tip cell behavior. (A–C) Representative examples of LECs sprouting from coated beads in control (A), VEGF-C (B)–, or anti-Nrp2B (C)–treated cultures stained with anti–LYVE-1. (D) Sprout number is significantly reduced with anti-Nrp2B or VEGFR3 ECD treatment compared with VEGF-C treatment alone. (E) Sprout length is not reduced with anti-Nrp2B treatment but is reduced by VEGFR3 ECD treatment compared with VEGF-C treatment alone (n = 5 beads/well for 10 wells). (F–I) Quantification of sprout initiation events (F), sprout-stalling events (G), and sprout extension rates from live imaging experiments. Error bars indicate SEM. *, P < 0.01. Bar, 150 µm.
Mentions: To directly evaluate the cellular basis for why Nrp2 inhibition affects sprout development, we tested the effects of anti-Nrp2B on LEC sprouting using an in vitro sprouting assay. Over the course of 14 d, the LECs form sprouts that radially extend from the bead and often display lumen formation and branching, which is highly reminiscent of lymphatic vessel sprouting in vivo. In the presence of VEGF-C, the LECs proliferate and form robust sprouts that extend from the bead (Fig. 4, A and B). Treatment with anti-Nrp2B reduced the number of sprouts (Fig. 4, C and D). Stimulation by VEGF-C resulted in an increase of sprout number from a mean of 2.4 sprouts per bead to a mean of 6.3 sprouts per bead (P < 0.01). Inhibition of Nrp2 by anti-Nrp2B in the presence of VEGF-C resulted in a significant reduction in the number of sprouts to 3.7 (P < 0.0001 compared with VEGF-C). As a positive control for blocking VEGF-C activity, the VEGFR3 extracellular domain protein (ECD; Mäkinen et al., 2001; Caunt et al., 2008) reduced sprouting to 1.7 sprouts per bead, which is a level comparable with the no VEGF-C control. Nrp2 inhibition did not affect mean sprout length (364 pixels in VEGF-C treated vs. 314 pixels in anti-Nrp2B treated; P = 0.09) in contrast to VEGFR3 ECD (53 pixels; P < 0.0001; Fig. 4 E). These data corroborate our in vivo observations that Nrp2 inhibition results in reduced sprouting but not sprout length, likely not affecting stalk cells. It also suggested an effect on the initial aspects of sprout formation.

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