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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 promotes FN-induced PXN Y118 phosphorylation. (A and B) HDMECs transiently transfected with control, NRP1 or VEGFR2 siRNA were plated on FN for the indicated times and immunofluorescently labeled (A) for pPXN Y118 (green) together with phalloidin (red) and DAPI (blue; bar, 20 µm) or immunoblotted for the indicated proteins (B). The single channel for pPXN staining is shown on the right side of A. (C) pPXN Y118 levels in immunoblots from 4 independent experiments were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SD in si-VEGFR2 or si-NRP1 relative to control siRNA transfected cells. *, P < 0.05, Student’s t test. (D) To examine if NRP1 forms a complex with pPXN in FN-stimulated ECs, HDMECs were detached in serum-free medium (nonadherent, NA) or plated on FN for the indicated times and then immunoprecipitated with control IgG or NRP1 antibodies, followed by immunoblotting for NRP1 and pPXN Y118. The immunoblot is representative of 4 independent experiments. (E) Single confocal scans of HDMECs plated on FN for 45 min and then immunofluorescently labeled with antibodies specific for human NRP1 or pPXN Y118 and counterstained with DAPI. Bar, 10 µm. A higher magnification of the area indicated with a dotted square is shown in the bottom row, and single channels for NRP1 and pPXN are shown adjacent to the triple stains at low and high magnification. Arrowheads indicate examples of partial colocalization. (F) HDMECs were transfected with pCDNA3.1 encoding wild-type mouse NRP1 or mutant mouse NRP1D320A, plated on FN for 60 min, and then labeled with antibodies specific for mouse NRP1 and pPXN and counterstained with DAPI. The single channel for pPXN staining is shown on the right. Cells expressing murine NRP1 (white arrowheads) up-regulate pPXN. Note that the antibody does not detect endogenous human NRP1 in untransfected cells (clear arrowheads). Bar, 20 µm.
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fig3: NRP1 promotes FN-induced PXN Y118 phosphorylation. (A and B) HDMECs transiently transfected with control, NRP1 or VEGFR2 siRNA were plated on FN for the indicated times and immunofluorescently labeled (A) for pPXN Y118 (green) together with phalloidin (red) and DAPI (blue; bar, 20 µm) or immunoblotted for the indicated proteins (B). The single channel for pPXN staining is shown on the right side of A. (C) pPXN Y118 levels in immunoblots from 4 independent experiments were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SD in si-VEGFR2 or si-NRP1 relative to control siRNA transfected cells. *, P < 0.05, Student’s t test. (D) To examine if NRP1 forms a complex with pPXN in FN-stimulated ECs, HDMECs were detached in serum-free medium (nonadherent, NA) or plated on FN for the indicated times and then immunoprecipitated with control IgG or NRP1 antibodies, followed by immunoblotting for NRP1 and pPXN Y118. The immunoblot is representative of 4 independent experiments. (E) Single confocal scans of HDMECs plated on FN for 45 min and then immunofluorescently labeled with antibodies specific for human NRP1 or pPXN Y118 and counterstained with DAPI. Bar, 10 µm. A higher magnification of the area indicated with a dotted square is shown in the bottom row, and single channels for NRP1 and pPXN are shown adjacent to the triple stains at low and high magnification. Arrowheads indicate examples of partial colocalization. (F) HDMECs were transfected with pCDNA3.1 encoding wild-type mouse NRP1 or mutant mouse NRP1D320A, plated on FN for 60 min, and then labeled with antibodies specific for mouse NRP1 and pPXN and counterstained with DAPI. The single channel for pPXN staining is shown on the right. Cells expressing murine NRP1 (white arrowheads) up-regulate pPXN. Note that the antibody does not detect endogenous human NRP1 in untransfected cells (clear arrowheads). Bar, 20 µm.

Mentions: Because pPXN is recruited to focal adhesions to promote their turnover during cell migration (Zaidel-Bar et al., 2007; Pasapera et al., 2010) and NRP1-deficient ECs have reduced pPXN (Figs. 1 and 2), we examined NRP1-dependent pPXN localization in FN-stimulated HDMECs by immunostaining. In control cells, pPXN increased over time and was present in focal adhesions at the end of F-actin stress fibers, correlating with an elongated cell shape (Fig. 3 A). FN-stimulated HDMECs, therefore, displayed the hallmarks of polarized cells. Although VEGFR2 down-regulation did not impair these responses, HDMECs lacking NRP1 displayed their characteristic rounded morphology with abundant cortical actin; correlating with the lack of stress fibers, pPXN levels were significantly decreased, with remaining pPXN being localized mainly to the cell periphery (Fig. 3, A and B). Immunoblotting established that siRNA targeting had been effective and further confirmed reduced pPXN in NRP1-deficient cells (Fig. 3 B); in contrast, pPXN was slightly but significantly increased in VEGFR2-deficient cells (Fig. 3, B and C). These findings demonstrate that NRP1 promotes PXN phosphorylation in a VEGFR2-independent fashion.


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 promotes FN-induced PXN Y118 phosphorylation. (A and B) HDMECs transiently transfected with control, NRP1 or VEGFR2 siRNA were plated on FN for the indicated times and immunofluorescently labeled (A) for pPXN Y118 (green) together with phalloidin (red) and DAPI (blue; bar, 20 µm) or immunoblotted for the indicated proteins (B). The single channel for pPXN staining is shown on the right side of A. (C) pPXN Y118 levels in immunoblots from 4 independent experiments were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SD in si-VEGFR2 or si-NRP1 relative to control siRNA transfected cells. *, P < 0.05, Student’s t test. (D) To examine if NRP1 forms a complex with pPXN in FN-stimulated ECs, HDMECs were detached in serum-free medium (nonadherent, NA) or plated on FN for the indicated times and then immunoprecipitated with control IgG or NRP1 antibodies, followed by immunoblotting for NRP1 and pPXN Y118. The immunoblot is representative of 4 independent experiments. (E) Single confocal scans of HDMECs plated on FN for 45 min and then immunofluorescently labeled with antibodies specific for human NRP1 or pPXN Y118 and counterstained with DAPI. Bar, 10 µm. A higher magnification of the area indicated with a dotted square is shown in the bottom row, and single channels for NRP1 and pPXN are shown adjacent to the triple stains at low and high magnification. Arrowheads indicate examples of partial colocalization. (F) HDMECs were transfected with pCDNA3.1 encoding wild-type mouse NRP1 or mutant mouse NRP1D320A, plated on FN for 60 min, and then labeled with antibodies specific for mouse NRP1 and pPXN and counterstained with DAPI. The single channel for pPXN staining is shown on the right. Cells expressing murine NRP1 (white arrowheads) up-regulate pPXN. Note that the antibody does not detect endogenous human NRP1 in untransfected cells (clear arrowheads). Bar, 20 µm.
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fig3: NRP1 promotes FN-induced PXN Y118 phosphorylation. (A and B) HDMECs transiently transfected with control, NRP1 or VEGFR2 siRNA were plated on FN for the indicated times and immunofluorescently labeled (A) for pPXN Y118 (green) together with phalloidin (red) and DAPI (blue; bar, 20 µm) or immunoblotted for the indicated proteins (B). The single channel for pPXN staining is shown on the right side of A. (C) pPXN Y118 levels in immunoblots from 4 independent experiments were quantified as pixel intensity relative to GAPDH and values expressed as mean fold change ± SD in si-VEGFR2 or si-NRP1 relative to control siRNA transfected cells. *, P < 0.05, Student’s t test. (D) To examine if NRP1 forms a complex with pPXN in FN-stimulated ECs, HDMECs were detached in serum-free medium (nonadherent, NA) or plated on FN for the indicated times and then immunoprecipitated with control IgG or NRP1 antibodies, followed by immunoblotting for NRP1 and pPXN Y118. The immunoblot is representative of 4 independent experiments. (E) Single confocal scans of HDMECs plated on FN for 45 min and then immunofluorescently labeled with antibodies specific for human NRP1 or pPXN Y118 and counterstained with DAPI. Bar, 10 µm. A higher magnification of the area indicated with a dotted square is shown in the bottom row, and single channels for NRP1 and pPXN are shown adjacent to the triple stains at low and high magnification. Arrowheads indicate examples of partial colocalization. (F) HDMECs were transfected with pCDNA3.1 encoding wild-type mouse NRP1 or mutant mouse NRP1D320A, plated on FN for 60 min, and then labeled with antibodies specific for mouse NRP1 and pPXN and counterstained with DAPI. The single channel for pPXN staining is shown on the right. Cells expressing murine NRP1 (white arrowheads) up-regulate pPXN. Note that the antibody does not detect endogenous human NRP1 in untransfected cells (clear arrowheads). Bar, 20 µm.
Mentions: Because pPXN is recruited to focal adhesions to promote their turnover during cell migration (Zaidel-Bar et al., 2007; Pasapera et al., 2010) and NRP1-deficient ECs have reduced pPXN (Figs. 1 and 2), we examined NRP1-dependent pPXN localization in FN-stimulated HDMECs by immunostaining. In control cells, pPXN increased over time and was present in focal adhesions at the end of F-actin stress fibers, correlating with an elongated cell shape (Fig. 3 A). FN-stimulated HDMECs, therefore, displayed the hallmarks of polarized cells. Although VEGFR2 down-regulation did not impair these responses, HDMECs lacking NRP1 displayed their characteristic rounded morphology with abundant cortical actin; correlating with the lack of stress fibers, pPXN levels were significantly decreased, with remaining pPXN being localized mainly to the cell periphery (Fig. 3, A and B). Immunoblotting established that siRNA targeting had been effective and further confirmed reduced pPXN in NRP1-deficient cells (Fig. 3 B); in contrast, pPXN was slightly but significantly increased in VEGFR2-deficient cells (Fig. 3, B and C). These findings demonstrate that NRP1 promotes PXN phosphorylation in a VEGFR2-independent fashion.

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