Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1-dependent ABL1 activation in endothelial cells.
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.
Affiliation: UCL Institute of Ophthalmology, University College London, London EC1V 9EL, England UK firstname.lastname@example.org email@example.com.
- Endothelial Cells/drug effects*/metabolism/physiology
- Neovascularization, Physiologic/drug effects*
- Proto-Oncogene Proteins c-abl/genetics/metabolism*
- Cell Adhesion/genetics
- Cell Movement/genetics
- Cells, Cultured
- Enzyme Activation/drug effects
- Imatinib Mesylate
- Mice, Inbred C57BL
- Mice, Knockout
- Mice, Transgenic
- Microscopy, Confocal
- Phosphorylation/drug effects
- Protein Kinase Inhibitors/pharmacology
- RNA Interference
- Retinal Neovascularization/genetics/physiopathology/prevention & control
- Vascular Endothelial Growth Factor A/metabolism
- Vascular Endothelial Growth Factor Receptor-2/genetics/metabolism
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fig7: ABL1 is essential for vessel spouting and branching in the retina. (A and B) P6 retinal vasculature of mice treated with vehicle or Imatinib from P2 to P5 was immunolabeled for IB4. Bar, 1 mm. Vascular extension from the retinal center to the vascular front is indicated with red arrows. (B) Vascular extension after Imatinib treatment was quantified as the distance of the IB4-positive front from the retinal center relative to the retinal radius (vehicle, n = 7 mice; Imatinib, n = 4 mice; **, P < 0.01, Student’s t test). Error bars show SD. (C–E) P6 retinal vasculature of mice treated with vehicle or Imatinib from P2 to P5 was immunolabeled for IB4 and FN. Bars: (C) 200 µm; (E) 50 µm. Note reduced FN staining of astrocyte processes ahead of the vascular front (Δ), whereas the vasculature was prominently stained for FN. (D) Quantification of FN pixel intensity in Imatinib-treated retinas in 0.06-mm2 areas of astrocyte networks ahead of the vascular front (fold change compared with controls; n = 3 mice each; *, P < 0.05, Student’s t test) and in vascular areas, isolated with an IB4-guided IMARIS mask (FN relative to IB4 pixel intensity; n = 3 mice each; P > 0.05, Student’s t test). Error bars show SD. Higher magnification of the areas indicated with dotted squares in C demonstrates abnormal filopodia and sprout morphology in Imatinib-treated retinas. The IB4 single channel is shown in grayscale below each panel after contrast enhancement to highlight filopodia. The arrow indicates an abnormally long and wide sprout without lateral protrusions or connections. Examples of abnormally thin, wavy, and misoriented filopodia are indicated with arrowheads. Note that the interaction of tip cells with microglia (wavy arrow) is not prevented by Imatinib treatment. (F and G) IB4-labeled P6 retinal vasculature of mice treated with vehicle or Imatinib by daily injections on P4 and P5 or from P2 to P5. Bar, 200 µm. Examples of abnormally long and wide sprouts without lateral protrusions or connections are indicated with arrows. (G) Quantification of filopodial bursts per vascular front length as an indicator of tip cell number, and quantification of branch points behind the vascular front (vehicle, n = 7 mice, vs. Imatinib P4-5, n = 3 mice, or Imatinib P2–5, n = 4 mice; ***, P < 0.001 vs. vehicle control, ANOVA with Tukey’s comparison test; #, P < 0.05 for P4–5 vs. P2–5 treatment, Student’s t test; P2–5 tamoxifen-injected Nrp1fl/fl mice lacking Cre, n = 8, or expressing Pdgfb-iCre-ERT2-Egfp, n = 6; *, P < 0.05; ***, P < 0.001 versus control, Student’s t test). Error bars show SD. (H) Schematic representation of the NRP1–ABL1–PXN pathway and its synergism with known VEGF signaling pathways transduced by VEGFR2.
Because ABL1 is involved in NRP1-mediated actin remodeling, cell migration, and PXN phosphorylation, we investigated next if it is also essential for blood vessel growth in vivo. IB4 labeling showed reduced vascular extension and network density in retinal flat mounts of P6 Imatinib-treated compared with vehicle-treated mice (Fig. 7 A). Quantitation confirmed a small but significant reduction in vascular extension across the retina (Fig. 7 B), as previously reported for loss of astrocyte FN (Stenzel et al., 2011). In agreement, FN immunostaining showed reduced astrocyte FN deposition ahead of the vascular front (Fig. 7, C and D), likely due to Imatinib targeting of PDGFR signaling in astrocytes (Buchdunger et al., 2002). In contrast, we observed abundant FN deposition around retinal vessels in both control and Imatinib-treated mice, with no difference in FN pixel intensity in IB4-positive areas (Fig. 7, C and D). Vascular FN assembly was therefore not compromised by ABL1 targeting.