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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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Abnormal lymphatic development in double-heterozygous nrp2+/−vegfr3+/− mice. (A) X-gal staining (blue) of E13.5 vegfr3+/− (left) and double-heterozygous nrp2+/−vegfr3+/− (right) littermate embryos. (B) expression levels of lymphatic marker genes as measured by quantitative PCR in RNA isolated from hearts of wild-type and double-heterozygote nrp2+/−vegfr2+/− and nrp2+/−vegfr3+/− mice. Values significantly different from wild-type mice (*, P > 0.05; **, P > 0.001) by Student’s t test are shown. Error bars indicate SEM. (C and D) Higher magnification of heads of embryos shown in A. Note numerous lymphatic vessel sprouts in vegfr3+/− (black arrows) and fewer enlarged lymph vessel sprouts in nrp2+/−vegfr3+/− (red arrows). e, eye; *, ear. (E and F) Transverse section through the neck of E13.5 embryos double stained with X-gal (blue) and CD-31 (brown). Note similar development of CD-31–positive arteries, veins, and skin capillaries (arrowheads) in vegfr3+/− (E) and nrp2+/−vegfr3+/− (F). Note enlarged jugular lymph sacs (asterisks) in nrp2+/−vegfr3+/− (F) compared with vegfr3+/− (E). Lymph vessels sprouting from the lymph sac toward the skin are less numerous in nrp2+/−vegfr3+/− compared with vegfr3+/− (arrows). A, arteries; V, veins; NT, neural tube; Vt, vertebra. Bars: (A) 1.4 mm; (C and D) 0.8 mm; (E and F) 150 µm.
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fig6: Abnormal lymphatic development in double-heterozygous nrp2+/−vegfr3+/− mice. (A) X-gal staining (blue) of E13.5 vegfr3+/− (left) and double-heterozygous nrp2+/−vegfr3+/− (right) littermate embryos. (B) expression levels of lymphatic marker genes as measured by quantitative PCR in RNA isolated from hearts of wild-type and double-heterozygote nrp2+/−vegfr2+/− and nrp2+/−vegfr3+/− mice. Values significantly different from wild-type mice (*, P > 0.05; **, P > 0.001) by Student’s t test are shown. Error bars indicate SEM. (C and D) Higher magnification of heads of embryos shown in A. Note numerous lymphatic vessel sprouts in vegfr3+/− (black arrows) and fewer enlarged lymph vessel sprouts in nrp2+/−vegfr3+/− (red arrows). e, eye; *, ear. (E and F) Transverse section through the neck of E13.5 embryos double stained with X-gal (blue) and CD-31 (brown). Note similar development of CD-31–positive arteries, veins, and skin capillaries (arrowheads) in vegfr3+/− (E) and nrp2+/−vegfr3+/− (F). Note enlarged jugular lymph sacs (asterisks) in nrp2+/−vegfr3+/− (F) compared with vegfr3+/− (E). Lymph vessels sprouting from the lymph sac toward the skin are less numerous in nrp2+/−vegfr3+/− compared with vegfr3+/− (arrows). A, arteries; V, veins; NT, neural tube; Vt, vertebra. Bars: (A) 1.4 mm; (C and D) 0.8 mm; (E and F) 150 µm.

Mentions: As the egfp insertion disrupts transcription of the vegfr2 gene, homozygous vegfr2egfp/egfp die at early embryonic stages as a result of failure of blood vessel formation. However, heterozygous mice are viable and fertile and develop no detectable blood vascular or lymphatic malformations. Double-heterozygous nrp2+/−vegfr2+/− mice were born at normal Mendelian ratios (12 litters, 79 mice, 16 wild type, 25 nrp2+/−, 16 vegfr2+/−, and 22 nrp2+/−vegfr2+/−). Real-time PCR of hearts isolated from P5 wild-type and nrp2+/−vegfr2+/− mice showed that, as expected, mRNA levels of flk-1/vegfr2 and nrp2 were decreased by ∼50% in the double-heterozygotes compared with wild-type littermates (Fig. 6 B). In contrast, levels of vegfr3 or podoplanin were not significantly different in nrp2+/−vegfr2+/− compared with wild type (Fig. 6 B).


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)

Abnormal lymphatic development in double-heterozygous nrp2+/−vegfr3+/− mice. (A) X-gal staining (blue) of E13.5 vegfr3+/− (left) and double-heterozygous nrp2+/−vegfr3+/− (right) littermate embryos. (B) expression levels of lymphatic marker genes as measured by quantitative PCR in RNA isolated from hearts of wild-type and double-heterozygote nrp2+/−vegfr2+/− and nrp2+/−vegfr3+/− mice. Values significantly different from wild-type mice (*, P > 0.05; **, P > 0.001) by Student’s t test are shown. Error bars indicate SEM. (C and D) Higher magnification of heads of embryos shown in A. Note numerous lymphatic vessel sprouts in vegfr3+/− (black arrows) and fewer enlarged lymph vessel sprouts in nrp2+/−vegfr3+/− (red arrows). e, eye; *, ear. (E and F) Transverse section through the neck of E13.5 embryos double stained with X-gal (blue) and CD-31 (brown). Note similar development of CD-31–positive arteries, veins, and skin capillaries (arrowheads) in vegfr3+/− (E) and nrp2+/−vegfr3+/− (F). Note enlarged jugular lymph sacs (asterisks) in nrp2+/−vegfr3+/− (F) compared with vegfr3+/− (E). Lymph vessels sprouting from the lymph sac toward the skin are less numerous in nrp2+/−vegfr3+/− compared with vegfr3+/− (arrows). A, arteries; V, veins; NT, neural tube; Vt, vertebra. Bars: (A) 1.4 mm; (C and D) 0.8 mm; (E and F) 150 µm.
© Copyright Policy - openaccess
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Show All Figures
getmorefigures.php?uid=PMC2812843&req=5

fig6: Abnormal lymphatic development in double-heterozygous nrp2+/−vegfr3+/− mice. (A) X-gal staining (blue) of E13.5 vegfr3+/− (left) and double-heterozygous nrp2+/−vegfr3+/− (right) littermate embryos. (B) expression levels of lymphatic marker genes as measured by quantitative PCR in RNA isolated from hearts of wild-type and double-heterozygote nrp2+/−vegfr2+/− and nrp2+/−vegfr3+/− mice. Values significantly different from wild-type mice (*, P > 0.05; **, P > 0.001) by Student’s t test are shown. Error bars indicate SEM. (C and D) Higher magnification of heads of embryos shown in A. Note numerous lymphatic vessel sprouts in vegfr3+/− (black arrows) and fewer enlarged lymph vessel sprouts in nrp2+/−vegfr3+/− (red arrows). e, eye; *, ear. (E and F) Transverse section through the neck of E13.5 embryos double stained with X-gal (blue) and CD-31 (brown). Note similar development of CD-31–positive arteries, veins, and skin capillaries (arrowheads) in vegfr3+/− (E) and nrp2+/−vegfr3+/− (F). Note enlarged jugular lymph sacs (asterisks) in nrp2+/−vegfr3+/− (F) compared with vegfr3+/− (E). Lymph vessels sprouting from the lymph sac toward the skin are less numerous in nrp2+/−vegfr3+/− compared with vegfr3+/− (arrows). A, arteries; V, veins; NT, neural tube; Vt, vertebra. Bars: (A) 1.4 mm; (C and D) 0.8 mm; (E and F) 150 µm.
Mentions: As the egfp insertion disrupts transcription of the vegfr2 gene, homozygous vegfr2egfp/egfp die at early embryonic stages as a result of failure of blood vessel formation. However, heterozygous mice are viable and fertile and develop no detectable blood vascular or lymphatic malformations. Double-heterozygous nrp2+/−vegfr2+/− mice were born at normal Mendelian ratios (12 litters, 79 mice, 16 wild type, 25 nrp2+/−, 16 vegfr2+/−, and 22 nrp2+/−vegfr2+/−). Real-time PCR of hearts isolated from P5 wild-type and nrp2+/−vegfr2+/− mice showed that, as expected, mRNA levels of flk-1/vegfr2 and nrp2 were decreased by ∼50% in the double-heterozygotes compared with wild-type littermates (Fig. 6 B). In contrast, levels of vegfr3 or podoplanin were not significantly different in nrp2+/−vegfr2+/− compared with wild type (Fig. 6 B).

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