Imatinib and endothelial NRP1 targeting inhibit pathological angiogenesis in mouse models of retinopathy and ear wound healing. (A) Immunostaining of a P17 wild-type mouse retina for FN together with IB4 after sequential exposure to 7 d of normoxia, 5 d of hyperoxia, and 5 d of normoxia. The area indicated with a square is shown in higher magnification adjacent to the first panel as double label and single channels. Bar, 1 mm. (B) Immunostaining for collagen IV together with IB4 of P17 retinas from mice treated with vehicle (control) or Imatinib by daily injections after return from hyperoxia to normoxia (P13–16). Pseudocoloring highlights AV (white) and neovascularization (NV, red). Bar, 1 mm. (C–F) Quantitation of AV and NV area in Imatinib-treated relative to vehicle-injected littermates (C and D; fold change; n = 8 retinas each) and in tamoxifen-injected Nrp1^fl/fl mice lacking or expressing Pdgfb-iCre-ERT2-Egfp (E and F; fold change; n ≥ 5 retinas each). Asterisks indicate statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001, Student’s t test. Error bars show SD. (G–J) To examine angiogenesis-dependent wound healing in the ear of vehicle- and Imatinib-treated mice between the day of injury (day 0, D0) and day 21, the punch wound was imaged on days 0 and 21 (G; bar, 2 mm) and immunostained for FN together with PECAM and DAPI in 40-µm ear sections on day 21 (H and I; bar, 200 µm). Higher magnifications of the areas indicated with dotted squares in H and I are shown in H′ and I′. Note the reduced number of blood vessels at the wound margin after Imatinib treatment (Δ). (J) Quantitation of wound diameter in vehicle and Imatinib-treated mice on days 0, 7, 14, and 21 after wounding (vehicle, n = 3 mice; Imatinib, n = 4 mice; *, P < 0.05; **, P < 0.01, Student’s t test for vehicle vs. Imatinib-treated mice at days 14 and 21). Error bars show SEM.