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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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ABL1 knockdown impairs PXN Y118 phosphorylation and EC migration. (A) qPCR analysis for Abl1 expression in HDMECs. Abl1 values were normalized to Actb and expressed as fold reduction in knockdown relative to control HDMECs (mean ± SD of 3 independent experiments). ***, P < 0.001, Student’s t test. (B–E) Immunofluorescence labeling (B and C) and immunoblotting (D and E) of HDMECs transfected with si-ABL1 or control siRNA and then plated on FN for the indicated times. In B, pPXN Y118 (green) is shown together with phalloidin (red) and DAPI (blue) on the left, and as single channel in grayscale on the right. Bars, 20 µm. The pixel intensity of the pPXN signal was quantified in C as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SEM of 4 independent experiments). *, P < 0.05, Student’s t test. (D and E) Immunoblotting shows that ABL1 down-regulation reduces PXN, but not ERK1/2 phosphorylation. pCRKL served as readout for ABL1 down-regulation and GAPDH as a loading control. The quantitation of pPXN Y118 levels as pixel intensity after densitometry is shown in E. Values are expressed as fold change in knockdown cells at the indicated time points relative to nonadherent (NA) control cells at 0 min (mean ± SD of 3 independent experiments). *, P < 0.05, Student’s t test. (F) HDMECs transfected with control or ABL1 siRNA were plated on FN-coated transwells and the percentage of transmigrated HDMECs determined after 240 min in knockdown relative to control cells (mean ± SEM in 4 independent experiments). **, P < 0.01, Student’s t test. (G and H) To investigate if ABL1 and NRP1 form a constitutive protein complex in ECs and associate with pPXN in FN-stimulated cells, HEK cells were transfected with expression vectors for NRP1 and ABL1 (+) before immunoprecipitation with control IgG or ABL1 antibody and immunoblotting for NRP1 (G), or immunoprecipitation with NRP1 and immunoblotting for ABL1 (H). Nontransfected cells (−) were used as internal negative control. (I) To examine complex formation of endogenous NRP1, ABL1, and pPXN, HDMECs were detached and lysed (nonadherent, NA) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with control IgG or ABL1 antibody followed by immunoblotting for NRP1 and pPXN Y118. (J and K) To examine if ABL1 recruits pPXN in a NRP1-dependent manner after FN stimulation, neonatal Nrp1fl/fl mice were induced with vehicle (control) or tamoxifen to delete NRP1 (J) and isolated MLECs cultured on FN before being detached and lysed (A) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with IgG or ABL1 antibody and immunoblotted for NRP1 and pPXN Y118 (K).
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fig4: ABL1 knockdown impairs PXN Y118 phosphorylation and EC migration. (A) qPCR analysis for Abl1 expression in HDMECs. Abl1 values were normalized to Actb and expressed as fold reduction in knockdown relative to control HDMECs (mean ± SD of 3 independent experiments). ***, P < 0.001, Student’s t test. (B–E) Immunofluorescence labeling (B and C) and immunoblotting (D and E) of HDMECs transfected with si-ABL1 or control siRNA and then plated on FN for the indicated times. In B, pPXN Y118 (green) is shown together with phalloidin (red) and DAPI (blue) on the left, and as single channel in grayscale on the right. Bars, 20 µm. The pixel intensity of the pPXN signal was quantified in C as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SEM of 4 independent experiments). *, P < 0.05, Student’s t test. (D and E) Immunoblotting shows that ABL1 down-regulation reduces PXN, but not ERK1/2 phosphorylation. pCRKL served as readout for ABL1 down-regulation and GAPDH as a loading control. The quantitation of pPXN Y118 levels as pixel intensity after densitometry is shown in E. Values are expressed as fold change in knockdown cells at the indicated time points relative to nonadherent (NA) control cells at 0 min (mean ± SD of 3 independent experiments). *, P < 0.05, Student’s t test. (F) HDMECs transfected with control or ABL1 siRNA were plated on FN-coated transwells and the percentage of transmigrated HDMECs determined after 240 min in knockdown relative to control cells (mean ± SEM in 4 independent experiments). **, P < 0.01, Student’s t test. (G and H) To investigate if ABL1 and NRP1 form a constitutive protein complex in ECs and associate with pPXN in FN-stimulated cells, HEK cells were transfected with expression vectors for NRP1 and ABL1 (+) before immunoprecipitation with control IgG or ABL1 antibody and immunoblotting for NRP1 (G), or immunoprecipitation with NRP1 and immunoblotting for ABL1 (H). Nontransfected cells (−) were used as internal negative control. (I) To examine complex formation of endogenous NRP1, ABL1, and pPXN, HDMECs were detached and lysed (nonadherent, NA) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with control IgG or ABL1 antibody followed by immunoblotting for NRP1 and pPXN Y118. (J and K) To examine if ABL1 recruits pPXN in a NRP1-dependent manner after FN stimulation, neonatal Nrp1fl/fl mice were induced with vehicle (control) or tamoxifen to delete NRP1 (J) and isolated MLECs cultured on FN before being detached and lysed (A) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with IgG or ABL1 antibody and immunoblotted for NRP1 and pPXN Y118 (K).

Mentions: Because NRP1 lacks catalytic activity, it requires a partner kinase to promote FN-induced PXN phosphorylation, but this kinase is not VEGFR2 (Fig. 1) or FAK (Fig. 2, B and C). A good candidate is the cell adhesion–associated kinase ABL1, which interacts with PXN in FN-stimulated fibroblasts (Lewis and Schwartz, 1998) as well as integrins β1 and β2 (Cui et al., 2009; Baruzzi et al., 2010). Furthermore, the Y118 residue that is phosphorylated in an NRP1-dependent fashion resides in an ABL1 phosphorylation consensus site (Cujec et al., 2002), and ABL1 is an effector of NRP1 and integrins in tumor matrix remodeling (Yaqoob et al., 2012). To investigate ABL1 function in FN-stimulated ECs, we used two independent but complementary methods: siRNA-mediated knockdown of ABL1 (Fig. 4) and pharmacological inhibition of ABL1 kinase activity (Fig. 5).


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)

ABL1 knockdown impairs PXN Y118 phosphorylation and EC migration. (A) qPCR analysis for Abl1 expression in HDMECs. Abl1 values were normalized to Actb and expressed as fold reduction in knockdown relative to control HDMECs (mean ± SD of 3 independent experiments). ***, P < 0.001, Student’s t test. (B–E) Immunofluorescence labeling (B and C) and immunoblotting (D and E) of HDMECs transfected with si-ABL1 or control siRNA and then plated on FN for the indicated times. In B, pPXN Y118 (green) is shown together with phalloidin (red) and DAPI (blue) on the left, and as single channel in grayscale on the right. Bars, 20 µm. The pixel intensity of the pPXN signal was quantified in C as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SEM of 4 independent experiments). *, P < 0.05, Student’s t test. (D and E) Immunoblotting shows that ABL1 down-regulation reduces PXN, but not ERK1/2 phosphorylation. pCRKL served as readout for ABL1 down-regulation and GAPDH as a loading control. The quantitation of pPXN Y118 levels as pixel intensity after densitometry is shown in E. Values are expressed as fold change in knockdown cells at the indicated time points relative to nonadherent (NA) control cells at 0 min (mean ± SD of 3 independent experiments). *, P < 0.05, Student’s t test. (F) HDMECs transfected with control or ABL1 siRNA were plated on FN-coated transwells and the percentage of transmigrated HDMECs determined after 240 min in knockdown relative to control cells (mean ± SEM in 4 independent experiments). **, P < 0.01, Student’s t test. (G and H) To investigate if ABL1 and NRP1 form a constitutive protein complex in ECs and associate with pPXN in FN-stimulated cells, HEK cells were transfected with expression vectors for NRP1 and ABL1 (+) before immunoprecipitation with control IgG or ABL1 antibody and immunoblotting for NRP1 (G), or immunoprecipitation with NRP1 and immunoblotting for ABL1 (H). Nontransfected cells (−) were used as internal negative control. (I) To examine complex formation of endogenous NRP1, ABL1, and pPXN, HDMECs were detached and lysed (nonadherent, NA) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with control IgG or ABL1 antibody followed by immunoblotting for NRP1 and pPXN Y118. (J and K) To examine if ABL1 recruits pPXN in a NRP1-dependent manner after FN stimulation, neonatal Nrp1fl/fl mice were induced with vehicle (control) or tamoxifen to delete NRP1 (J) and isolated MLECs cultured on FN before being detached and lysed (A) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with IgG or ABL1 antibody and immunoblotted for NRP1 and pPXN Y118 (K).
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fig4: ABL1 knockdown impairs PXN Y118 phosphorylation and EC migration. (A) qPCR analysis for Abl1 expression in HDMECs. Abl1 values were normalized to Actb and expressed as fold reduction in knockdown relative to control HDMECs (mean ± SD of 3 independent experiments). ***, P < 0.001, Student’s t test. (B–E) Immunofluorescence labeling (B and C) and immunoblotting (D and E) of HDMECs transfected with si-ABL1 or control siRNA and then plated on FN for the indicated times. In B, pPXN Y118 (green) is shown together with phalloidin (red) and DAPI (blue) on the left, and as single channel in grayscale on the right. Bars, 20 µm. The pixel intensity of the pPXN signal was quantified in C as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SEM of 4 independent experiments). *, P < 0.05, Student’s t test. (D and E) Immunoblotting shows that ABL1 down-regulation reduces PXN, but not ERK1/2 phosphorylation. pCRKL served as readout for ABL1 down-regulation and GAPDH as a loading control. The quantitation of pPXN Y118 levels as pixel intensity after densitometry is shown in E. Values are expressed as fold change in knockdown cells at the indicated time points relative to nonadherent (NA) control cells at 0 min (mean ± SD of 3 independent experiments). *, P < 0.05, Student’s t test. (F) HDMECs transfected with control or ABL1 siRNA were plated on FN-coated transwells and the percentage of transmigrated HDMECs determined after 240 min in knockdown relative to control cells (mean ± SEM in 4 independent experiments). **, P < 0.01, Student’s t test. (G and H) To investigate if ABL1 and NRP1 form a constitutive protein complex in ECs and associate with pPXN in FN-stimulated cells, HEK cells were transfected with expression vectors for NRP1 and ABL1 (+) before immunoprecipitation with control IgG or ABL1 antibody and immunoblotting for NRP1 (G), or immunoprecipitation with NRP1 and immunoblotting for ABL1 (H). Nontransfected cells (−) were used as internal negative control. (I) To examine complex formation of endogenous NRP1, ABL1, and pPXN, HDMECs were detached and lysed (nonadherent, NA) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with control IgG or ABL1 antibody followed by immunoblotting for NRP1 and pPXN Y118. (J and K) To examine if ABL1 recruits pPXN in a NRP1-dependent manner after FN stimulation, neonatal Nrp1fl/fl mice were induced with vehicle (control) or tamoxifen to delete NRP1 (J) and isolated MLECs cultured on FN before being detached and lysed (A) or lysed after plating on FN for the indicated times. Lysates were immunoprecipitated with IgG or ABL1 antibody and immunoblotted for NRP1 and pPXN Y118 (K).
Mentions: Because NRP1 lacks catalytic activity, it requires a partner kinase to promote FN-induced PXN phosphorylation, but this kinase is not VEGFR2 (Fig. 1) or FAK (Fig. 2, B and C). A good candidate is the cell adhesion–associated kinase ABL1, which interacts with PXN in FN-stimulated fibroblasts (Lewis and Schwartz, 1998) as well as integrins β1 and β2 (Cui et al., 2009; Baruzzi et al., 2010). Furthermore, the Y118 residue that is phosphorylated in an NRP1-dependent fashion resides in an ABL1 phosphorylation consensus site (Cujec et al., 2002), and ABL1 is an effector of NRP1 and integrins in tumor matrix remodeling (Yaqoob et al., 2012). To investigate ABL1 function in FN-stimulated ECs, we used two independent but complementary methods: siRNA-mediated knockdown of ABL1 (Fig. 4) and pharmacological inhibition of ABL1 kinase activity (Fig. 5).

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