Limits...
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.

Show MeSH

Related in: MedlinePlus

The Nrp1VEGF− mutant mice exhibit normal gross morphology.(A) Whole-mount images of the heart at P9 show the normal cardiac morphology of the Nrp1VEGF− mutants. (B and C) Organ weights measured at P9 (B) and adulthood (C) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in Nrp1VEGF− animals, n ≥ 5. (D) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in Nrp1VEGF− animals. (E) Viability table depicts the predicted and observed frequencies for each genotype at the indicated developmental stages. The table values represent the percentage of the total number of animals genotyped per age while the total number of animals is shown in parentheses.DOI:http://dx.doi.org/10.7554/eLife.03720.009
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4197402&req=5

fig3s2: The Nrp1VEGF− mutant mice exhibit normal gross morphology.(A) Whole-mount images of the heart at P9 show the normal cardiac morphology of the Nrp1VEGF− mutants. (B and C) Organ weights measured at P9 (B) and adulthood (C) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in Nrp1VEGF− animals, n ≥ 5. (D) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in Nrp1VEGF− animals. (E) Viability table depicts the predicted and observed frequencies for each genotype at the indicated developmental stages. The table values represent the percentage of the total number of animals genotyped per age while the total number of animals is shown in parentheses.DOI:http://dx.doi.org/10.7554/eLife.03720.009

Mentions: A gene replacement strategy was implemented to generate a mouse line harboring the Nrp1D320K mutation in the endogenous Nrp1 locus, delineated as Nrp1VEGF−. Specifically, two base pair mutations were introduced into exon 6 of the mouse Nrp1 gene to produce the D320K mutation in the endogenous Asp320 location (Figure 3A). After recombineering, embryonic stem cells were screened via PCR and sequenced to confirm that the D320K mutation was appropriately introduced into the Nrp1 locus (Figure 3—figure supplement 1A–C). Once Nrp1VEGF− mice were obtained, the presence of the D320K mutation was verified by sequencing (Figure 3—figure supplement 1D). Importantly, the Nrp1VEGF− mutants expressed normal levels of NRP1 protein as assessed by Western blot on embryonic day 14.5 (E14.5) lung and adult heart, brain, lung and kidney (Figure 3C, Figure 3—figure supplement 2D). AP-VEGF and AP-SEMA3A binding was examined at E12.5 in the dorsal root entry zone (DREZ), where NRP1-expressing axons from the dorsal root ganglion enter the spinal cord. Both AP-VEGF and AP-SEMA3A bound to the DREZ in control animals (Figure 3B) while AP-VEGF binding to the DREZ was abolished in the Nrp1VEGF− mutant (Figure 3B), confirming that this mutation eliminated VEGF-NRP1 binding in vivo. Moreover, NRP1 immunostaining and AP-SEMA3A binding to the DREZ appeared similar between Nrp1VEGF− and control littermates (Figure 3B). Finally, the Nrp1VEGF− mutants failed to display the perinatal lethality or cardiac defect observed in the Nrp1Sema− mutants (Gu et al., 2003), further confirming functional SEMA3-NRP1 binding in Nrp1VEGF− mice (Figure 3—figure supplement 1).10.7554/eLife.03720.007Figure 3.Nrp1VEGF- mice selectively abolish VEGF-NRP1 binding in vivo.


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 exhibit normal gross morphology.(A) Whole-mount images of the heart at P9 show the normal cardiac morphology of the Nrp1VEGF− mutants. (B and C) Organ weights measured at P9 (B) and adulthood (C) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in Nrp1VEGF− animals, n ≥ 5. (D) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in Nrp1VEGF− animals. (E) Viability table depicts the predicted and observed frequencies for each genotype at the indicated developmental stages. The table values represent the percentage of the total number of animals genotyped per age while the total number of animals is shown in parentheses.DOI:http://dx.doi.org/10.7554/eLife.03720.009
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3s2: The Nrp1VEGF− mutant mice exhibit normal gross morphology.(A) Whole-mount images of the heart at P9 show the normal cardiac morphology of the Nrp1VEGF− mutants. (B and C) Organ weights measured at P9 (B) and adulthood (C) demonstrate that the heart, brain, lung, and kidney undergo appropriate growth in Nrp1VEGF− animals, n ≥ 5. (D) Western blots from adult heart, brain, lung, and kidney tissue demonstrate that NRP1 protein levels were not affected in Nrp1VEGF− animals. (E) Viability table depicts the predicted and observed frequencies for each genotype at the indicated developmental stages. The table values represent the percentage of the total number of animals genotyped per age while the total number of animals is shown in parentheses.DOI:http://dx.doi.org/10.7554/eLife.03720.009
Mentions: A gene replacement strategy was implemented to generate a mouse line harboring the Nrp1D320K mutation in the endogenous Nrp1 locus, delineated as Nrp1VEGF−. Specifically, two base pair mutations were introduced into exon 6 of the mouse Nrp1 gene to produce the D320K mutation in the endogenous Asp320 location (Figure 3A). After recombineering, embryonic stem cells were screened via PCR and sequenced to confirm that the D320K mutation was appropriately introduced into the Nrp1 locus (Figure 3—figure supplement 1A–C). Once Nrp1VEGF− mice were obtained, the presence of the D320K mutation was verified by sequencing (Figure 3—figure supplement 1D). Importantly, the Nrp1VEGF− mutants expressed normal levels of NRP1 protein as assessed by Western blot on embryonic day 14.5 (E14.5) lung and adult heart, brain, lung and kidney (Figure 3C, Figure 3—figure supplement 2D). AP-VEGF and AP-SEMA3A binding was examined at E12.5 in the dorsal root entry zone (DREZ), where NRP1-expressing axons from the dorsal root ganglion enter the spinal cord. Both AP-VEGF and AP-SEMA3A bound to the DREZ in control animals (Figure 3B) while AP-VEGF binding to the DREZ was abolished in the Nrp1VEGF− mutant (Figure 3B), confirming that this mutation eliminated VEGF-NRP1 binding in vivo. Moreover, NRP1 immunostaining and AP-SEMA3A binding to the DREZ appeared similar between Nrp1VEGF− and control littermates (Figure 3B). Finally, the Nrp1VEGF− mutants failed to display the perinatal lethality or cardiac defect observed in the Nrp1Sema− mutants (Gu et al., 2003), further confirming functional SEMA3-NRP1 binding in Nrp1VEGF− mice (Figure 3—figure supplement 1).10.7554/eLife.03720.007Figure 3.Nrp1VEGF- mice selectively abolish VEGF-NRP1 binding in vivo.

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