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Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.

Raimondi C, Fantin A, Lampropoulou A, Denti L, Chikh A, Ruhrberg C - J. Exp. Med. (2014)

Bottom Line: NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling.Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins.The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England UK c.raimondi@ucl.ac.uk c.ruhrberg@ucl.ac.uk.

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NRP1 transduces ECM signals independently of VEGF165 and VEGFR2. (A–C) For phosphoproteomic screening, HDMECs were transfected with control or NRP1 siRNA before immunoblotting to confirm NRP1 knockdown (A) or stimulation with VEGF165 for 10 min (B) or FN for 30 min (C), followed by phosphokinase-antibody array screening. Phosphorylation of the indicated proteins in si-NRP1 relative to si-control transfected cells from 2 independent experiments, performed in duplicate each, were shown as mean fold change ± SEM. (D and E) To validate NRP1-regulated phosphoproteins identified in the phosphoproteomic screen, HDMECs were transiently transfected with control, NRP1 or VEGFR2 siRNA and stimulated with VEGF165 (D) or plated on FN (E) for the indicated times. Lysates were immunoblotted for the indicated proteins, with GAPDH serving as a loading control. Immunoblots are representative of 3 independent experiments. (F) pERK (T202/Y204) levels were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SEM. *, P < 0.05, Student’s t test.
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fig2: NRP1 transduces ECM signals independently of VEGF165 and VEGFR2. (A–C) For phosphoproteomic screening, HDMECs were transfected with control or NRP1 siRNA before immunoblotting to confirm NRP1 knockdown (A) or stimulation with VEGF165 for 10 min (B) or FN for 30 min (C), followed by phosphokinase-antibody array screening. Phosphorylation of the indicated proteins in si-NRP1 relative to si-control transfected cells from 2 independent experiments, performed in duplicate each, were shown as mean fold change ± SEM. (D and E) To validate NRP1-regulated phosphoproteins identified in the phosphoproteomic screen, HDMECs were transiently transfected with control, NRP1 or VEGFR2 siRNA and stimulated with VEGF165 (D) or plated on FN (E) for the indicated times. Lysates were immunoblotted for the indicated proteins, with GAPDH serving as a loading control. Immunoblots are representative of 3 independent experiments. (F) pERK (T202/Y204) levels were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SEM. *, P < 0.05, Student’s t test.

Mentions: Because FN stimulation activated NRP1-dependent actin remodeling and cell migration independently of VEGF165 and VEGFR2, we sought to identify candidate effectors with a phosphokinase antibody array (e.g., Zhuang et al., 2012). For this experiment, HDMECs were transfected with si-NRP1 or control siRNA (Fig. 2 A), and then serum-starved and stimulated with VEGF165 (Fig. 2 B) or plated on FN (Fig. 2 C). We observed reduced VEGF165-induced activation of the MAPK p38 in ECs lacking NRP1, as expected (Kawamura et al., 2008), and additionally impaired FN-induced P38 activation in ECs lacking NRP1 (Fig. 2, B and C). The screen also showed that NRP1 down-regulation in VEGF165-stimulated HDMECs reduced AKT activation (Fig. 2 B), as previously reported for HUVECs (Pan et al., 2007a,b; Koch et al., 2011). Unexpectedly, however, NRP1 down-regulation did not impair AKT phosphorylation in FN-stimulated ECs (Fig. 2 C).


Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.

Raimondi C, Fantin A, Lampropoulou A, Denti L, Chikh A, Ruhrberg C - J. Exp. Med. (2014)

NRP1 transduces ECM signals independently of VEGF165 and VEGFR2. (A–C) For phosphoproteomic screening, HDMECs were transfected with control or NRP1 siRNA before immunoblotting to confirm NRP1 knockdown (A) or stimulation with VEGF165 for 10 min (B) or FN for 30 min (C), followed by phosphokinase-antibody array screening. Phosphorylation of the indicated proteins in si-NRP1 relative to si-control transfected cells from 2 independent experiments, performed in duplicate each, were shown as mean fold change ± SEM. (D and E) To validate NRP1-regulated phosphoproteins identified in the phosphoproteomic screen, HDMECs were transiently transfected with control, NRP1 or VEGFR2 siRNA and stimulated with VEGF165 (D) or plated on FN (E) for the indicated times. Lysates were immunoblotted for the indicated proteins, with GAPDH serving as a loading control. Immunoblots are representative of 3 independent experiments. (F) pERK (T202/Y204) levels were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SEM. *, P < 0.05, Student’s t test.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042645&req=5

fig2: NRP1 transduces ECM signals independently of VEGF165 and VEGFR2. (A–C) For phosphoproteomic screening, HDMECs were transfected with control or NRP1 siRNA before immunoblotting to confirm NRP1 knockdown (A) or stimulation with VEGF165 for 10 min (B) or FN for 30 min (C), followed by phosphokinase-antibody array screening. Phosphorylation of the indicated proteins in si-NRP1 relative to si-control transfected cells from 2 independent experiments, performed in duplicate each, were shown as mean fold change ± SEM. (D and E) To validate NRP1-regulated phosphoproteins identified in the phosphoproteomic screen, HDMECs were transiently transfected with control, NRP1 or VEGFR2 siRNA and stimulated with VEGF165 (D) or plated on FN (E) for the indicated times. Lysates were immunoblotted for the indicated proteins, with GAPDH serving as a loading control. Immunoblots are representative of 3 independent experiments. (F) pERK (T202/Y204) levels were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SEM. *, P < 0.05, Student’s t test.
Mentions: Because FN stimulation activated NRP1-dependent actin remodeling and cell migration independently of VEGF165 and VEGFR2, we sought to identify candidate effectors with a phosphokinase antibody array (e.g., Zhuang et al., 2012). For this experiment, HDMECs were transfected with si-NRP1 or control siRNA (Fig. 2 A), and then serum-starved and stimulated with VEGF165 (Fig. 2 B) or plated on FN (Fig. 2 C). We observed reduced VEGF165-induced activation of the MAPK p38 in ECs lacking NRP1, as expected (Kawamura et al., 2008), and additionally impaired FN-induced P38 activation in ECs lacking NRP1 (Fig. 2, B and C). The screen also showed that NRP1 down-regulation in VEGF165-stimulated HDMECs reduced AKT activation (Fig. 2 B), as previously reported for HUVECs (Pan et al., 2007a,b; Koch et al., 2011). Unexpectedly, however, NRP1 down-regulation did not impair AKT phosphorylation in FN-stimulated ECs (Fig. 2 C).

Bottom Line: NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling.Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins.The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth.

View Article: PubMed Central - HTML - PubMed

Affiliation: UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England UK c.raimondi@ucl.ac.uk c.ruhrberg@ucl.ac.uk.

Show MeSH
Related in: MedlinePlus