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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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The Nrp1VEGF− mutant mice display normal vessel branching and coverage at postnatal stages.(A) Vessel staining with isolectin (green) demonstrates that the Nrp1VEGF− mutants have normal vessel coverage and branching in the cerebral cortex at P7. (B and C) Quantification of vessel coverage and branching in P7 cortex shown in A, n = 3. Scale bar: 200 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.011
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fig4s1: The Nrp1VEGF− mutant mice display normal vessel branching and coverage at postnatal stages.(A) Vessel staining with isolectin (green) demonstrates that the Nrp1VEGF− mutants have normal vessel coverage and branching in the cerebral cortex at P7. (B and C) Quantification of vessel coverage and branching in P7 cortex shown in A, n = 3. Scale bar: 200 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.011

Mentions: To thoroughly examine vascular integrity during development, isolectin staining was employed to visualize blood vessels in embryonic and perinatal brain sections and vessel ingression, morphology, and branching were assessed in the Nrp1VEGF− mutant. Surprisingly, Nrp1VEGF− animals did not exhibit any of the vascular abnormalities observed in the endothelial-specific NRP1 knock-out. As shown in Figure 4A and quantified in Figure 4B–C, cortical vessel ingression was nearly absent in Tie2-Cre;Nrp1fl/fl animals at E11.5 while ingression was unaffected in the Nrp1VEGF− mutants. In addition, Tie2-Cre;Nrp1fl/fl animals had abnormally large vascular aggregates distributed throughout the striatum at E14.5 while vessels were evenly dispersed without aggregates in both control and Nrp1VEGF− animals (Figure 4D–F). Finally, Tie2-Cre;Nrp1fl/fl animals had a significant decrease in vessel branching in the cortex at E14.5 while Nrp1VEGF− animals exhibited normal vessel branching (Figure 4G–I). Moreover, unlike the endothelial-specific NRP1 knock-out, the long term viability of the Nrp1VEGF− mutants allowed us to assess cortical vessel branching and coverage at P7 which was indistinguishable from control littermates (Figure 4G–I, Figure 4—figure supplement 1). Therefore, VEGF-NRP1 binding is not required for developmental angiogenesis.10.7554/eLife.03720.010Figure 4.VEGF-NRP1 binding is not required for developmental angiogenesis.


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)

The Nrp1VEGF− mutant mice display normal vessel branching and coverage at postnatal stages.(A) Vessel staining with isolectin (green) demonstrates that the Nrp1VEGF− mutants have normal vessel coverage and branching in the cerebral cortex at P7. (B and C) Quantification of vessel coverage and branching in P7 cortex shown in A, n = 3. Scale bar: 200 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.011
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4s1: The Nrp1VEGF− mutant mice display normal vessel branching and coverage at postnatal stages.(A) Vessel staining with isolectin (green) demonstrates that the Nrp1VEGF− mutants have normal vessel coverage and branching in the cerebral cortex at P7. (B and C) Quantification of vessel coverage and branching in P7 cortex shown in A, n = 3. Scale bar: 200 μm.DOI:http://dx.doi.org/10.7554/eLife.03720.011
Mentions: To thoroughly examine vascular integrity during development, isolectin staining was employed to visualize blood vessels in embryonic and perinatal brain sections and vessel ingression, morphology, and branching were assessed in the Nrp1VEGF− mutant. Surprisingly, Nrp1VEGF− animals did not exhibit any of the vascular abnormalities observed in the endothelial-specific NRP1 knock-out. As shown in Figure 4A and quantified in Figure 4B–C, cortical vessel ingression was nearly absent in Tie2-Cre;Nrp1fl/fl animals at E11.5 while ingression was unaffected in the Nrp1VEGF− mutants. In addition, Tie2-Cre;Nrp1fl/fl animals had abnormally large vascular aggregates distributed throughout the striatum at E14.5 while vessels were evenly dispersed without aggregates in both control and Nrp1VEGF− animals (Figure 4D–F). Finally, Tie2-Cre;Nrp1fl/fl animals had a significant decrease in vessel branching in the cortex at E14.5 while Nrp1VEGF− animals exhibited normal vessel branching (Figure 4G–I). Moreover, unlike the endothelial-specific NRP1 knock-out, the long term viability of the Nrp1VEGF− mutants allowed us to assess cortical vessel branching and coverage at P7 which was indistinguishable from control littermates (Figure 4G–I, Figure 4—figure supplement 1). Therefore, VEGF-NRP1 binding is not required for developmental angiogenesis.10.7554/eLife.03720.010Figure 4.VEGF-NRP1 binding is not required for developmental angiogenesis.

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