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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 is dispensable for cell matrix adhesion but promotes FN-induced cell spreading, filopodia extension, and motility of primary ECs. (A–C) Immunoblotting with the indicated antibodies to demonstrate NRP1 knockdown (A) and time course of cell adhesion (B and C) in HDMECs and HUVECs transfected with control or NRP1 siRNA and plated for the indicated times on plastic dishes coated with 10 µg/ml FN (mean ± SEM of 3 independent experiments). (D–H) HDMECs transfected with control, VEGFR2 or NRP1 siRNA were plated on FN for the indicated times before immunoblotting (D), qPCR analysis (E) or fluorescent labeling (F) with the F-actin marker phalloidin (green) and the nuclear counterstain DAPI (blue). Bar, 20 µm. Images shown in the right two columns are higher magnifications of areas indicated with dotted squares. Also shown is a quantification of cell area (G) and phalloidin-stained microspikes (H) at the indicated time points (mean ± SEM of ≥30 cells from 3 independent experiments). (I) HDMECs transfected with si-NRP1 were plated on FN in the presence of 50 ng/ml VEGF165 for the indicated times before fluorescent labeling with phalloidin (green) and DAPI (blue). Bar, 20 µm. (J and K) HDMECs transfected with control or NRP1 siRNAs were plated on FN and cell motility on FN observed for 200 min. J shows representative tracks, with the point of origin of each cell plotted as 0 at the axis intersection. K shows mean track length (mean ± SEM of 23 cells from 2 independent experiments). (L–N) HDMECs or HUVECs transfected with control, VEGFR2 or NRP1 siRNA (L) and Nrp1fl/fl MLECs transfected with adenovirus carrying Gfp control or Cre transgenes (M and N) were plated on FN-coated transwells. The number of transmigrated cells was determined after 240 min (mean ± SEM from ≥3 experiments, each performed in duplicate). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.
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fig1: NRP1 is dispensable for cell matrix adhesion but promotes FN-induced cell spreading, filopodia extension, and motility of primary ECs. (A–C) Immunoblotting with the indicated antibodies to demonstrate NRP1 knockdown (A) and time course of cell adhesion (B and C) in HDMECs and HUVECs transfected with control or NRP1 siRNA and plated for the indicated times on plastic dishes coated with 10 µg/ml FN (mean ± SEM of 3 independent experiments). (D–H) HDMECs transfected with control, VEGFR2 or NRP1 siRNA were plated on FN for the indicated times before immunoblotting (D), qPCR analysis (E) or fluorescent labeling (F) with the F-actin marker phalloidin (green) and the nuclear counterstain DAPI (blue). Bar, 20 µm. Images shown in the right two columns are higher magnifications of areas indicated with dotted squares. Also shown is a quantification of cell area (G) and phalloidin-stained microspikes (H) at the indicated time points (mean ± SEM of ≥30 cells from 3 independent experiments). (I) HDMECs transfected with si-NRP1 were plated on FN in the presence of 50 ng/ml VEGF165 for the indicated times before fluorescent labeling with phalloidin (green) and DAPI (blue). Bar, 20 µm. (J and K) HDMECs transfected with control or NRP1 siRNAs were plated on FN and cell motility on FN observed for 200 min. J shows representative tracks, with the point of origin of each cell plotted as 0 at the axis intersection. K shows mean track length (mean ± SEM of 23 cells from 2 independent experiments). (L–N) HDMECs or HUVECs transfected with control, VEGFR2 or NRP1 siRNA (L) and Nrp1fl/fl MLECs transfected with adenovirus carrying Gfp control or Cre transgenes (M and N) were plated on FN-coated transwells. The number of transmigrated cells was determined after 240 min (mean ± SEM from ≥3 experiments, each performed in duplicate). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.

Mentions: Because NRP1 is known to interact with FN-binding integrins (Fukasawa et al., 2007; Robinson et al., 2009; Valdembri et al., 2009), we first examined the requirement of NRP1 for EC adhesion, spreading, and actin remodeling. For these experiments, we used human dermal microvascular ECs (HDMECs) because dermal vasculature naturally undergoes extensive angiogenesis during wound healing. We additionally used human umbilical vein ECs (HUVECs), as they have been widely used to study VEGF-induced signaling mechanisms (e.g., Soker et al., 2002; Pan et al., 2007a; Chen et al., 2010). We transfected primary cells with a previously validated siRNA that targets NRP1 or a control nonsense siRNA. Having confirmed the efficacy of this approach (Fig. 1 A), we tested HDMEC and HUVEC adhesion to tissue culture dishes coated with 10 µg/ml FN, a concentration which effectively promotes cell adhesion and migration (Clark et al., 1986; Sottile et al., 1998; Tvorogov et al., 2005). In contrast to earlier studies, which reported that NRP1 loss inhibits HUVEC adhesion at FN concentrations <5 µg/ml (Murga et al., 2005; Valdembri et al., 2009), we found that adhesion was not compromised by NRP1 deficiency in either HDMECs or HUVECs at 10 µg/ml FN (Fig. 1, B and C). We had therefore identified conditions suitable to study FN-induced cell spreading, actin remodeling, and cell migration of ECs in the absence of prior defects in cell attachment.


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 is dispensable for cell matrix adhesion but promotes FN-induced cell spreading, filopodia extension, and motility of primary ECs. (A–C) Immunoblotting with the indicated antibodies to demonstrate NRP1 knockdown (A) and time course of cell adhesion (B and C) in HDMECs and HUVECs transfected with control or NRP1 siRNA and plated for the indicated times on plastic dishes coated with 10 µg/ml FN (mean ± SEM of 3 independent experiments). (D–H) HDMECs transfected with control, VEGFR2 or NRP1 siRNA were plated on FN for the indicated times before immunoblotting (D), qPCR analysis (E) or fluorescent labeling (F) with the F-actin marker phalloidin (green) and the nuclear counterstain DAPI (blue). Bar, 20 µm. Images shown in the right two columns are higher magnifications of areas indicated with dotted squares. Also shown is a quantification of cell area (G) and phalloidin-stained microspikes (H) at the indicated time points (mean ± SEM of ≥30 cells from 3 independent experiments). (I) HDMECs transfected with si-NRP1 were plated on FN in the presence of 50 ng/ml VEGF165 for the indicated times before fluorescent labeling with phalloidin (green) and DAPI (blue). Bar, 20 µm. (J and K) HDMECs transfected with control or NRP1 siRNAs were plated on FN and cell motility on FN observed for 200 min. J shows representative tracks, with the point of origin of each cell plotted as 0 at the axis intersection. K shows mean track length (mean ± SEM of 23 cells from 2 independent experiments). (L–N) HDMECs or HUVECs transfected with control, VEGFR2 or NRP1 siRNA (L) and Nrp1fl/fl MLECs transfected with adenovirus carrying Gfp control or Cre transgenes (M and N) were plated on FN-coated transwells. The number of transmigrated cells was determined after 240 min (mean ± SEM from ≥3 experiments, each performed in duplicate). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.
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fig1: NRP1 is dispensable for cell matrix adhesion but promotes FN-induced cell spreading, filopodia extension, and motility of primary ECs. (A–C) Immunoblotting with the indicated antibodies to demonstrate NRP1 knockdown (A) and time course of cell adhesion (B and C) in HDMECs and HUVECs transfected with control or NRP1 siRNA and plated for the indicated times on plastic dishes coated with 10 µg/ml FN (mean ± SEM of 3 independent experiments). (D–H) HDMECs transfected with control, VEGFR2 or NRP1 siRNA were plated on FN for the indicated times before immunoblotting (D), qPCR analysis (E) or fluorescent labeling (F) with the F-actin marker phalloidin (green) and the nuclear counterstain DAPI (blue). Bar, 20 µm. Images shown in the right two columns are higher magnifications of areas indicated with dotted squares. Also shown is a quantification of cell area (G) and phalloidin-stained microspikes (H) at the indicated time points (mean ± SEM of ≥30 cells from 3 independent experiments). (I) HDMECs transfected with si-NRP1 were plated on FN in the presence of 50 ng/ml VEGF165 for the indicated times before fluorescent labeling with phalloidin (green) and DAPI (blue). Bar, 20 µm. (J and K) HDMECs transfected with control or NRP1 siRNAs were plated on FN and cell motility on FN observed for 200 min. J shows representative tracks, with the point of origin of each cell plotted as 0 at the axis intersection. K shows mean track length (mean ± SEM of 23 cells from 2 independent experiments). (L–N) HDMECs or HUVECs transfected with control, VEGFR2 or NRP1 siRNA (L) and Nrp1fl/fl MLECs transfected with adenovirus carrying Gfp control or Cre transgenes (M and N) were plated on FN-coated transwells. The number of transmigrated cells was determined after 240 min (mean ± SEM from ≥3 experiments, each performed in duplicate). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test.
Mentions: Because NRP1 is known to interact with FN-binding integrins (Fukasawa et al., 2007; Robinson et al., 2009; Valdembri et al., 2009), we first examined the requirement of NRP1 for EC adhesion, spreading, and actin remodeling. For these experiments, we used human dermal microvascular ECs (HDMECs) because dermal vasculature naturally undergoes extensive angiogenesis during wound healing. We additionally used human umbilical vein ECs (HUVECs), as they have been widely used to study VEGF-induced signaling mechanisms (e.g., Soker et al., 2002; Pan et al., 2007a; Chen et al., 2010). We transfected primary cells with a previously validated siRNA that targets NRP1 or a control nonsense siRNA. Having confirmed the efficacy of this approach (Fig. 1 A), we tested HDMEC and HUVEC adhesion to tissue culture dishes coated with 10 µg/ml FN, a concentration which effectively promotes cell adhesion and migration (Clark et al., 1986; Sottile et al., 1998; Tvorogov et al., 2005). In contrast to earlier studies, which reported that NRP1 loss inhibits HUVEC adhesion at FN concentrations <5 µg/ml (Murga et al., 2005; Valdembri et al., 2009), we found that adhesion was not compromised by NRP1 deficiency in either HDMECs or HUVECs at 10 µg/ml FN (Fig. 1, B and C). We had therefore identified conditions suitable to study FN-induced cell spreading, actin remodeling, and cell migration of ECs in the absence of prior defects in cell attachment.

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