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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 and ABL1 promote PXN phosphorylation during retinal angiogenesis in the mouse. (A) A schematic representation of retinal angiogenesis illustrates how vessels (red) expand from the retinal center toward the periphery (indicated by arrows). Vessels are guided by an astrocyte-derived FN network (green) ahead of the vascular front, but FN subsequently overlaps partially with the vessel pattern (red with green outline). (B) Immunolabeling for FN together with the vascular marker IB4 demonstrates angiogenesis in the P6 retina. Bar, 400 µm. The area shown in higher magnification is indicated with a dotted square in the first panel. (C) Reduced NRP1 levels in Nrp1+/− compared with Nrp1+/+ littermates, shown by immunoblot quantification of NRP1 protein levels in P6 lungs. NRP1 levels were normalized to VE-cadherin levels and expressed as percentage relative to wild type (mean ± SEM; n ≥ 3). *, P < 0.05, Student’s t test. (D–H) Single confocal slices through the vascular front in retinas of Nrp1+/+, Nrp1+/− littermate mice and tamoxifen-inducible EC-specific Nrp1- mice injected with tamoxifen from P2 to P5 (D) or treated with vehicle or 100 mg/kg/d Imatinib on days P4 and P5 (G). Retinas were immunostained for pPXN Y118 and IB4. Bar, 15 µm. In D and G (right), Imaris software was used to mask areas not labeled for IB4 to reveal pPXN staining in ECs. Quantification of vascular pPXN in optical z-stacks after applying a mask to isolate IB4-positive areas in Nrp1+/− (E), tamoxifen-inducible EC-specific Nrp1- mutants (F), and Imatinib-treated mice (H) relative to controls. Mean pixel values of pPXN relative to IB4 are expressed as percentage of control ± SEM (n = 3). *, P < 0.05, Student’s t test.
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fig6: NRP1 and ABL1 promote PXN phosphorylation during retinal angiogenesis in the mouse. (A) A schematic representation of retinal angiogenesis illustrates how vessels (red) expand from the retinal center toward the periphery (indicated by arrows). Vessels are guided by an astrocyte-derived FN network (green) ahead of the vascular front, but FN subsequently overlaps partially with the vessel pattern (red with green outline). (B) Immunolabeling for FN together with the vascular marker IB4 demonstrates angiogenesis in the P6 retina. Bar, 400 µm. The area shown in higher magnification is indicated with a dotted square in the first panel. (C) Reduced NRP1 levels in Nrp1+/− compared with Nrp1+/+ littermates, shown by immunoblot quantification of NRP1 protein levels in P6 lungs. NRP1 levels were normalized to VE-cadherin levels and expressed as percentage relative to wild type (mean ± SEM; n ≥ 3). *, P < 0.05, Student’s t test. (D–H) Single confocal slices through the vascular front in retinas of Nrp1+/+, Nrp1+/− littermate mice and tamoxifen-inducible EC-specific Nrp1- mice injected with tamoxifen from P2 to P5 (D) or treated with vehicle or 100 mg/kg/d Imatinib on days P4 and P5 (G). Retinas were immunostained for pPXN Y118 and IB4. Bar, 15 µm. In D and G (right), Imaris software was used to mask areas not labeled for IB4 to reveal pPXN staining in ECs. Quantification of vascular pPXN in optical z-stacks after applying a mask to isolate IB4-positive areas in Nrp1+/− (E), tamoxifen-inducible EC-specific Nrp1- mutants (F), and Imatinib-treated mice (H) relative to controls. Mean pixel values of pPXN relative to IB4 are expressed as percentage of control ± SEM (n = 3). *, P < 0.05, Student’s t test.

Mentions: To examine if NRP1 and ABL1 also promote PXN phosphorylation in an ECM-dependent angiogenesis model in vivo, we studied the perinatal mouse retina; in this organ, endothelial sprouts headed by filopodia-studded tip cells migrate toward astrocyte-localized VEGF in the retinal periphery, with filopodia being guided by astrocyte-derived FN (Fig. 6 A; Ruhrberg et al., 2002; Gerhardt et al., 2003; Stenzel et al., 2011). Immunostaining confirmed FN deposition around IB4-stained vessels and ahead of the vascular front in a fine meshwork characteristic of astrocytes (Fig. 6 B). To investigate how NRP1 deficiency affected pPXN levels during retinal angiogenesis, we could not use Nrp1−/− mice, as they die before birth (Kawasaki et al., 1999). Instead, we compared Nrp1+/+ and Nrp1+/− littermates because the latter are viable but nevertheless have mild angiogenesis defects (Fantin et al., 2013). Immunoblotting confirmed significantly decreased NRP1 levels in P6 Nrp1+/− mice (Fig. 6 C). Single optical slices, acquired by confocal microscopy after immunolabeling, revealed pPXN staining in ECs at the IB4-positive vascular front, including tip cells and their filopodia, and also some pPXN ahead of the vascular front (Fig. 6 D). As observed for si-NRP1–targeted HDMECs, pPXN staining appeared reduced in Nrp1+/− littermates, both in vascular and avascular areas (Fig. 6 D). To confirm that this defect was cell autonomous in vascular endothelium, we also used Cre-LoxP recombination approach to delete NRP1 (Fig. 1). Thus, conditional Nrp1- mice lacking or expressing a tamoxifen-inducible, endothelial-specific Cre transgene were treated with tamoxifen from postnatal day 2 (P2) to P5 and stained for IB4 and pPXN. As seen in Nrp1+/− mice, tamoxifen-treatment reduced pPXN in Cre-expressing but not Cre-negative (control) littermates (Fig. 6 D).


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 and ABL1 promote PXN phosphorylation during retinal angiogenesis in the mouse. (A) A schematic representation of retinal angiogenesis illustrates how vessels (red) expand from the retinal center toward the periphery (indicated by arrows). Vessels are guided by an astrocyte-derived FN network (green) ahead of the vascular front, but FN subsequently overlaps partially with the vessel pattern (red with green outline). (B) Immunolabeling for FN together with the vascular marker IB4 demonstrates angiogenesis in the P6 retina. Bar, 400 µm. The area shown in higher magnification is indicated with a dotted square in the first panel. (C) Reduced NRP1 levels in Nrp1+/− compared with Nrp1+/+ littermates, shown by immunoblot quantification of NRP1 protein levels in P6 lungs. NRP1 levels were normalized to VE-cadherin levels and expressed as percentage relative to wild type (mean ± SEM; n ≥ 3). *, P < 0.05, Student’s t test. (D–H) Single confocal slices through the vascular front in retinas of Nrp1+/+, Nrp1+/− littermate mice and tamoxifen-inducible EC-specific Nrp1- mice injected with tamoxifen from P2 to P5 (D) or treated with vehicle or 100 mg/kg/d Imatinib on days P4 and P5 (G). Retinas were immunostained for pPXN Y118 and IB4. Bar, 15 µm. In D and G (right), Imaris software was used to mask areas not labeled for IB4 to reveal pPXN staining in ECs. Quantification of vascular pPXN in optical z-stacks after applying a mask to isolate IB4-positive areas in Nrp1+/− (E), tamoxifen-inducible EC-specific Nrp1- mutants (F), and Imatinib-treated mice (H) relative to controls. Mean pixel values of pPXN relative to IB4 are expressed as percentage of control ± SEM (n = 3). *, P < 0.05, Student’s t test.
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fig6: NRP1 and ABL1 promote PXN phosphorylation during retinal angiogenesis in the mouse. (A) A schematic representation of retinal angiogenesis illustrates how vessels (red) expand from the retinal center toward the periphery (indicated by arrows). Vessels are guided by an astrocyte-derived FN network (green) ahead of the vascular front, but FN subsequently overlaps partially with the vessel pattern (red with green outline). (B) Immunolabeling for FN together with the vascular marker IB4 demonstrates angiogenesis in the P6 retina. Bar, 400 µm. The area shown in higher magnification is indicated with a dotted square in the first panel. (C) Reduced NRP1 levels in Nrp1+/− compared with Nrp1+/+ littermates, shown by immunoblot quantification of NRP1 protein levels in P6 lungs. NRP1 levels were normalized to VE-cadherin levels and expressed as percentage relative to wild type (mean ± SEM; n ≥ 3). *, P < 0.05, Student’s t test. (D–H) Single confocal slices through the vascular front in retinas of Nrp1+/+, Nrp1+/− littermate mice and tamoxifen-inducible EC-specific Nrp1- mice injected with tamoxifen from P2 to P5 (D) or treated with vehicle or 100 mg/kg/d Imatinib on days P4 and P5 (G). Retinas were immunostained for pPXN Y118 and IB4. Bar, 15 µm. In D and G (right), Imaris software was used to mask areas not labeled for IB4 to reveal pPXN staining in ECs. Quantification of vascular pPXN in optical z-stacks after applying a mask to isolate IB4-positive areas in Nrp1+/− (E), tamoxifen-inducible EC-specific Nrp1- mutants (F), and Imatinib-treated mice (H) relative to controls. Mean pixel values of pPXN relative to IB4 are expressed as percentage of control ± SEM (n = 3). *, P < 0.05, Student’s t test.
Mentions: To examine if NRP1 and ABL1 also promote PXN phosphorylation in an ECM-dependent angiogenesis model in vivo, we studied the perinatal mouse retina; in this organ, endothelial sprouts headed by filopodia-studded tip cells migrate toward astrocyte-localized VEGF in the retinal periphery, with filopodia being guided by astrocyte-derived FN (Fig. 6 A; Ruhrberg et al., 2002; Gerhardt et al., 2003; Stenzel et al., 2011). Immunostaining confirmed FN deposition around IB4-stained vessels and ahead of the vascular front in a fine meshwork characteristic of astrocytes (Fig. 6 B). To investigate how NRP1 deficiency affected pPXN levels during retinal angiogenesis, we could not use Nrp1−/− mice, as they die before birth (Kawasaki et al., 1999). Instead, we compared Nrp1+/+ and Nrp1+/− littermates because the latter are viable but nevertheless have mild angiogenesis defects (Fantin et al., 2013). Immunoblotting confirmed significantly decreased NRP1 levels in P6 Nrp1+/− mice (Fig. 6 C). Single optical slices, acquired by confocal microscopy after immunolabeling, revealed pPXN staining in ECs at the IB4-positive vascular front, including tip cells and their filopodia, and also some pPXN ahead of the vascular front (Fig. 6 D). As observed for si-NRP1–targeted HDMECs, pPXN staining appeared reduced in Nrp1+/− littermates, both in vascular and avascular areas (Fig. 6 D). To confirm that this defect was cell autonomous in vascular endothelium, we also used Cre-LoxP recombination approach to delete NRP1 (Fig. 1). Thus, conditional Nrp1- mice lacking or expressing a tamoxifen-inducible, endothelial-specific Cre transgene were treated with tamoxifen from postnatal day 2 (P2) to P5 and stained for IB4 and pPXN. As seen in Nrp1+/− mice, tamoxifen-treatment reduced pPXN in Cre-expressing but not Cre-negative (control) littermates (Fig. 6 D).

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