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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: 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.

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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VEGF-NRP1 binding is not required for NRP1-VEGFR2 complex formation in vitro and in vivo.(A) HEK293T cells transfected with Vegfr2 and either WT Nrp1 or Nrp1D230K exhibited normal NRP1-VEGFR2 complex formation. (B) Lung lysates generated from the Nrp1VEGF− mutants also displayed normal NRP1-VEGFR2 complex formation comparable to littermate controls.DOI:http://dx.doi.org/10.7554/eLife.03720.013
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fig5s1: VEGF-NRP1 binding is not required for NRP1-VEGFR2 complex formation in vitro and in vivo.(A) HEK293T cells transfected with Vegfr2 and either WT Nrp1 or Nrp1D230K exhibited normal NRP1-VEGFR2 complex formation. (B) Lung lysates generated from the Nrp1VEGF− mutants also displayed normal NRP1-VEGFR2 complex formation comparable to littermate controls.DOI:http://dx.doi.org/10.7554/eLife.03720.013

Mentions: To test this second possibility, VEGFR2 expression was evaluated in the Tie2-Cre;Nrp1fl/− mutants and control littermates via Western blot on E14.5 lung tissue. This biochemical assay revealed that total VEGFR2 protein levels were significantly reduced in the Tie2-Cre;Nrp1fl/− mutants compared to their control littermates (Figure 5A–B). To determine the cell surface expression of VEGFR2 in vivo, we used fluorescence-activated cell sorting (FACS) to specifically quantify VEGFR2 expression at the cell surface of non-permeabilized endothelial cells derived from the acutely dissociated lungs of Tie2-Cre;Nrp1fl/− and control embryos. Remarkably, Tie2-Cre;Nrp1fl/− mutants displayed a significant decrease in the fluorescence intensity of VEGFR2 labeling as compared to control littermates (Figure 5E–F), suggesting that NRP1 functions to regulate VEGFR2 surface expression in endothelial cells. In contrast, both Western blot and FACS analysis determined that VEGFR2 protein levels were unperturbed in Nrp1VEGF− animals (Figure 5C–D,G–F). In addition, co-immunoprecipitation on P7 lung tissue revealed that NRP1 and VEGFR2 are physically associated in both control and Nrp1VEGF− animals (Figure 5—figure supplement 1B), validating that NRP1-VEGFR2 receptor complex formation does not require VEGF-NRP1 binding in vivo. This result mimics our co-immunoprecipitation experiments on HEK293T cells transfected with either WT Nrp1 or Nrp1D320K constructs (Figure 5—figure supplement 1A). Together, these findings indicate that NRP1 plays a role in regulating the cell surface expression of VEGFR2 in endothelial cells and that VEGF-NRP1 binding is not necessary for this function in vivo (Figure 5G).10.7554/eLife.03720.012Figure 5.NRP1 regulates VEGFR2 expression at the cell surface independent of VEGF-NRP1 binding.


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-NRP1 binding is not required for NRP1-VEGFR2 complex formation in vitro and in vivo.(A) HEK293T cells transfected with Vegfr2 and either WT Nrp1 or Nrp1D230K exhibited normal NRP1-VEGFR2 complex formation. (B) Lung lysates generated from the Nrp1VEGF− mutants also displayed normal NRP1-VEGFR2 complex formation comparable to littermate controls.DOI:http://dx.doi.org/10.7554/eLife.03720.013
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5s1: VEGF-NRP1 binding is not required for NRP1-VEGFR2 complex formation in vitro and in vivo.(A) HEK293T cells transfected with Vegfr2 and either WT Nrp1 or Nrp1D230K exhibited normal NRP1-VEGFR2 complex formation. (B) Lung lysates generated from the Nrp1VEGF− mutants also displayed normal NRP1-VEGFR2 complex formation comparable to littermate controls.DOI:http://dx.doi.org/10.7554/eLife.03720.013
Mentions: To test this second possibility, VEGFR2 expression was evaluated in the Tie2-Cre;Nrp1fl/− mutants and control littermates via Western blot on E14.5 lung tissue. This biochemical assay revealed that total VEGFR2 protein levels were significantly reduced in the Tie2-Cre;Nrp1fl/− mutants compared to their control littermates (Figure 5A–B). To determine the cell surface expression of VEGFR2 in vivo, we used fluorescence-activated cell sorting (FACS) to specifically quantify VEGFR2 expression at the cell surface of non-permeabilized endothelial cells derived from the acutely dissociated lungs of Tie2-Cre;Nrp1fl/− and control embryos. Remarkably, Tie2-Cre;Nrp1fl/− mutants displayed a significant decrease in the fluorescence intensity of VEGFR2 labeling as compared to control littermates (Figure 5E–F), suggesting that NRP1 functions to regulate VEGFR2 surface expression in endothelial cells. In contrast, both Western blot and FACS analysis determined that VEGFR2 protein levels were unperturbed in Nrp1VEGF− animals (Figure 5C–D,G–F). In addition, co-immunoprecipitation on P7 lung tissue revealed that NRP1 and VEGFR2 are physically associated in both control and Nrp1VEGF− animals (Figure 5—figure supplement 1B), validating that NRP1-VEGFR2 receptor complex formation does not require VEGF-NRP1 binding in vivo. This result mimics our co-immunoprecipitation experiments on HEK293T cells transfected with either WT Nrp1 or Nrp1D320K constructs (Figure 5—figure supplement 1A). Together, these findings indicate that NRP1 plays a role in regulating the cell surface expression of VEGFR2 in endothelial cells and that VEGF-NRP1 binding is not necessary for this function in vivo (Figure 5G).10.7554/eLife.03720.012Figure 5.NRP1 regulates VEGFR2 expression at the cell surface independent of VEGF-NRP1 binding.

Bottom Line: 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.

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