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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.

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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 kinase activity is essential for PXN Y118 phosphorylation in HDMECs in vitro. (A–C) To examine if ABL1 kinase activity is essential for PXN phosphorylation, we performed immunofluorescence labeling (A and B) and immunoblotting (C) of HDMECs treated with vehicle or Imatinib 30 min before and during plating on FN for the indicated times. In A, 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. pPXN pixel intensity was quantified in B and expressed as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SD of 4 independent experiments). *, P < 0.05, Student’s t test. (C) Immunoblotting confirmed that Imatinib treatment reduced pPXN Y118 levels. Total ERK1/2 levels were used as a loading control. (D) To examine if endogenous NRP1 and ABL1 form a complex in ECs, we performed coimmunoprecipitation of endogenous proteins from lysates of HDMECs, treated with vehicle or 10 µM Imatinib for 30 min, detached, and plated on FN for the indicated times in the presence of Imatinib. Immunoprecipitation using ABL1 antibody was followed by immunoblotting performed for NRP1 and ABL1.
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fig5: ABL1 kinase activity is essential for PXN Y118 phosphorylation in HDMECs in vitro. (A–C) To examine if ABL1 kinase activity is essential for PXN phosphorylation, we performed immunofluorescence labeling (A and B) and immunoblotting (C) of HDMECs treated with vehicle or Imatinib 30 min before and during plating on FN for the indicated times. In A, 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. pPXN pixel intensity was quantified in B and expressed as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SD of 4 independent experiments). *, P < 0.05, Student’s t test. (C) Immunoblotting confirmed that Imatinib treatment reduced pPXN Y118 levels. Total ERK1/2 levels were used as a loading control. (D) To examine if endogenous NRP1 and ABL1 form a complex in ECs, we performed coimmunoprecipitation of endogenous proteins from lysates of HDMECs, treated with vehicle or 10 µM Imatinib for 30 min, detached, and plated on FN for the indicated times in the presence of Imatinib. Immunoprecipitation using ABL1 antibody was followed by immunoblotting performed for NRP1 and ABL1.

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 kinase activity is essential for PXN Y118 phosphorylation in HDMECs in vitro. (A–C) To examine if ABL1 kinase activity is essential for PXN phosphorylation, we performed immunofluorescence labeling (A and B) and immunoblotting (C) of HDMECs treated with vehicle or Imatinib 30 min before and during plating on FN for the indicated times. In A, 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. pPXN pixel intensity was quantified in B and expressed as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SD of 4 independent experiments). *, P < 0.05, Student’s t test. (C) Immunoblotting confirmed that Imatinib treatment reduced pPXN Y118 levels. Total ERK1/2 levels were used as a loading control. (D) To examine if endogenous NRP1 and ABL1 form a complex in ECs, we performed coimmunoprecipitation of endogenous proteins from lysates of HDMECs, treated with vehicle or 10 µM Imatinib for 30 min, detached, and plated on FN for the indicated times in the presence of Imatinib. Immunoprecipitation using ABL1 antibody was followed by immunoblotting performed for NRP1 and ABL1.
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fig5: ABL1 kinase activity is essential for PXN Y118 phosphorylation in HDMECs in vitro. (A–C) To examine if ABL1 kinase activity is essential for PXN phosphorylation, we performed immunofluorescence labeling (A and B) and immunoblotting (C) of HDMECs treated with vehicle or Imatinib 30 min before and during plating on FN for the indicated times. In A, 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. pPXN pixel intensity was quantified in B and expressed as fold change in knockdown cells at the indicated time points relative to control cells at 60 min (mean ± SD of 4 independent experiments). *, P < 0.05, Student’s t test. (C) Immunoblotting confirmed that Imatinib treatment reduced pPXN Y118 levels. Total ERK1/2 levels were used as a loading control. (D) To examine if endogenous NRP1 and ABL1 form a complex in ECs, we performed coimmunoprecipitation of endogenous proteins from lysates of HDMECs, treated with vehicle or 10 µM Imatinib for 30 min, detached, and plated on FN for the indicated times in the presence of Imatinib. Immunoprecipitation using ABL1 antibody was followed by immunoblotting performed for NRP1 and ABL1.
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