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Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

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Related in: MedlinePlus

VEGF-induced VEGFR2 phosphorylation is reduced in both the Nrp1VEGF− and Tie2-Cre;Nrp1fl/− mutants.(A) Western blot from E14.5 lung tissue shows that VEGFR2 phosphorylation upon VEGF treatment was diminished in the Nrp1VEGF− mutant. (B) Quantification of VEGFR2 phosphorylation shown in A, n = 7. (C) Western blot from E14.5 lung tissue demonstrates that VEGFR2 phosphorylation is significantly reduced in the Tie2-Cre;Nrp1fl/− mutants. (D) Quantification of VEGFR2 phosphorylation shown in B, n = 5.DOI:http://dx.doi.org/10.7554/eLife.03720.014
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fig5s2: VEGF-induced VEGFR2 phosphorylation is reduced in both the Nrp1VEGF− and Tie2-Cre;Nrp1fl/− mutants.(A) Western blot from E14.5 lung tissue shows that VEGFR2 phosphorylation upon VEGF treatment was diminished in the Nrp1VEGF− mutant. (B) Quantification of VEGFR2 phosphorylation shown in A, n = 7. (C) Western blot from E14.5 lung tissue demonstrates that VEGFR2 phosphorylation is significantly reduced in the Tie2-Cre;Nrp1fl/− mutants. (D) Quantification of VEGFR2 phosphorylation shown in B, n = 5.DOI:http://dx.doi.org/10.7554/eLife.03720.014

Mentions: To examine VEGF signaling in the Tie2-Cre;Nrp1fl/− and Nrp1VEGF− mutants, VEGFR2 phosphorylation was examined via Western blot on embryonic lung tissue isolated at E14.5. Specifically, Tie2-Cre;Nrp1fl/− mutants had a severe reduction in VEGFR2 phosphorylation at the tyrosine residue 1175 (Y1175) upon VEGF treatment (Figure 5—figure supplement 2A,B). Interestingly, Nrp1VEGF− mutants also exhibited a mild reduction in VEGFR2 phosphorylation while total VEGFR2 protein levels were well maintained (Figure 5—figure supplement 2C,D). Although the level of pVEGFR2 in the Nrp1VEGF− mutant was sufficiently high to support vascular development during embryogenesis, the modest reduction in pVEGFR2 may manifest in issues with angiogenesis, vascular maintenance, and regeneration in the postnatal animal.


Neuropilin-1 functions as a VEGFR2 co-receptor to guide developmental angiogenesis independent of ligand binding.

Gelfand MV, Hagan N, Tata A, Oh WJ, Lacoste B, Kang KT, Kopycinska J, Bischoff J, Wang JH, Gu C - Elife (2014)

VEGF-induced VEGFR2 phosphorylation is reduced in both the Nrp1VEGF− and Tie2-Cre;Nrp1fl/− mutants.(A) Western blot from E14.5 lung tissue shows that VEGFR2 phosphorylation upon VEGF treatment was diminished in the Nrp1VEGF− mutant. (B) Quantification of VEGFR2 phosphorylation shown in A, n = 7. (C) Western blot from E14.5 lung tissue demonstrates that VEGFR2 phosphorylation is significantly reduced in the Tie2-Cre;Nrp1fl/− mutants. (D) Quantification of VEGFR2 phosphorylation shown in B, n = 5.DOI:http://dx.doi.org/10.7554/eLife.03720.014
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197402&req=5

fig5s2: VEGF-induced VEGFR2 phosphorylation is reduced in both the Nrp1VEGF− and Tie2-Cre;Nrp1fl/− mutants.(A) Western blot from E14.5 lung tissue shows that VEGFR2 phosphorylation upon VEGF treatment was diminished in the Nrp1VEGF− mutant. (B) Quantification of VEGFR2 phosphorylation shown in A, n = 7. (C) Western blot from E14.5 lung tissue demonstrates that VEGFR2 phosphorylation is significantly reduced in the Tie2-Cre;Nrp1fl/− mutants. (D) Quantification of VEGFR2 phosphorylation shown in B, n = 5.DOI:http://dx.doi.org/10.7554/eLife.03720.014
Mentions: To examine VEGF signaling in the Tie2-Cre;Nrp1fl/− and Nrp1VEGF− mutants, VEGFR2 phosphorylation was examined via Western blot on embryonic lung tissue isolated at E14.5. Specifically, Tie2-Cre;Nrp1fl/− mutants had a severe reduction in VEGFR2 phosphorylation at the tyrosine residue 1175 (Y1175) upon VEGF treatment (Figure 5—figure supplement 2A,B). Interestingly, Nrp1VEGF− mutants also exhibited a mild reduction in VEGFR2 phosphorylation while total VEGFR2 protein levels were well maintained (Figure 5—figure supplement 2C,D). Although the level of pVEGFR2 in the Nrp1VEGF− mutant was sufficiently high to support vascular development during embryogenesis, the modest reduction in pVEGFR2 may manifest in issues with angiogenesis, vascular maintenance, and regeneration in the postnatal animal.

Bottom Line: In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)).Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2.Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, Harvard Medical School, Boston, United States.

ABSTRACT
During development, tissue repair, and tumor growth, most blood vessel networks are generated through angiogenesis. Vascular endothelial growth factor (VEGF) is a key regulator of this process and currently both VEGF and its receptors, VEGFR1, VEGFR2, and Neuropilin1 (NRP1), are targeted in therapeutic strategies for vascular disease and cancer. NRP1 is essential for vascular morphogenesis, but how NRP1 functions to guide vascular development has not been completely elucidated. In this study, we generated a mouse line harboring a point mutation in the endogenous Nrp1 locus that selectively abolishes VEGF-NRP1 binding (Nrp1(VEGF-)). Nrp1(VEGF-) mutants survive to adulthood with normal vasculature revealing that NRP1 functions independent of VEGF-NRP1 binding during developmental angiogenesis. Moreover, we found that Nrp1-deficient vessels have reduced VEGFR2 surface expression in vivo demonstrating that NRP1 regulates its co-receptor, VEGFR2. Given the resources invested in NRP1-targeted anti-angiogenesis therapies, our results will be integral for developing strategies to re-build vasculature in disease.

Show MeSH
Related in: MedlinePlus