Volume 4, Issue 6, Pages 915-924 (December 1999) Selective Requirement for Src Kinases during VEGF-Induced Angiogenesis and Vascular Permeability  Brian P Eliceiri, Robert Paul, Pamela L Schwartzberg, John D Hood, Jie Leng, David A Cheresh  Molecular Cell  Volume 4, Issue 6, Pages 915-924 (December 1999) DOI: 10.1016/S1097-2765(00)80221-X

Figure 1 Activation of Endogenous Src Kinase Activity by bFGF and VEGF and the Effect of Kinase-Deleted Src on Angiogenesis In Vivo (A) Tissue extracts of 10-day-old chick CAMs were exposed to filter paper disks saturated with bFGF or VEGF (2 μg/ml) for 2 hr. Endogenous Src was immunoprecipitated from equivalent amounts of total protein and subjected to an in vitro immune complex kinase assays with a FAK–GST fusion protein as a substrate, electrophoresed, and transferred to nitrocellulose. The relative fold increase in Src activity is indicated in italics. The above kinase assay blot was probed with an anti-Src antibody as a loading control for equivalent Src and IgG content. (B) Chick CAMs (9 day) were exposed to filter paper disks saturated with RCAS–Src 251 (kinase deleted) or RCAS–GFP containing retroviruses or buffer for 20 hr and then incubated in the presence or absence of bFGF or VEGF for an additional 72 hr. Tissue extracts of these CAMs were examined for endogenous Src activity by in vitro immune complex kinase assay as described above using FAK–GST as a substrate. (C) The level of angiogenesis was quantified in embryos incubated with RCAS–Src251 or RCAS–GFP followed by stimulation with either bFGF or VEGF as described above. Blood vessels were enumerated by counting blood vessel branch points in a double blinded manner. Each bar represents the mean ± SEM of three replicates. (D) Micrographs of representative CAMs were taken with an Olympus stereomicroscope. Scale bar, 350 μm. Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)

Figure 2 Retroviral Delivery of RCAS–Src 251 to Human Tumors Growing on the Chick CAM Reverses Tumor Growth (A) Human DAOY medulloblastomas, which express VEGF, were grown on the CAM of chick embryos as described in the Experimental Procedures. Retrovirus containing RCAS–GFP or RCAS–Src 251 was topically applied to preestablished tumors of greater than 50 mg. A representative micrograph of a medulloblastoma tumor fragment infected with RCAS–GFP expressing GFP reveals exclusive expression in the tumor blood vessels (arrowhead) as detected by optical sectioning with a Bio-Rad 1024 laser confocal scanning microscope. Scale bar, 500 μm. (B) Tumors treated as above were allowed to grow for 3 or 6 days, after which they were resected and wet weights were determined. Data are expressed as the mean change in tumor weight (from the 50 mg tumor starting weight) ± SEM of two replicates. RCAS–Src 251 had a significant impact on tumor growth after 3 days (*p < 0.002) and 6 days (**p < 0.05). (C) Representative stereomicrographs of medulloblastoma tumors surgically removed from the embryos were taken with an Olympus stereomicroscope (scale bar, 350 μm). (Lower panel) A high magnification micrograph of each tumor showing the vasculature in detail (scale bar, 350 μm). The arrowhead indicates blood vessel disruption in RCAS–Src 251–treated tumors. Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)

Figure 3 Apoptosis in VEGF-Stimulated Blood Vessels Expressing Src 251 (A) Immunolocalization of factor VIII–related antigen (von Willlebrand factor), apoptag immunostaining of apoptotic cells, and nuclear staining with DAPI in cryosections of CAMs expressing RCAS–Src 251 or RCAS–GFP, after stimulation with bFGF or VEGF as described in Figure 1. The merge represents an overlay of the factor VIII staining and apoptag staining. The fluorescence from the GFP was not preserved in the fixation protocol used for the indirect immunofluorescence in these experiments. These micrographs were representative of blood vessel staining in duplicate samples. Scale bar, 50 μm. (B) Apoptotic cells were identified by annexin V staining of RCAS–Src 251–infected CAMS treated with VEGF and detected by flow cytometry. Collagenase-dissociated cells isolated from RCAS–Src 251– (black) or RCAS-GFP- (mock, white) infected CAMs treated with VEGF, as described in Figure 1, were incubated with annexin V. The fluorescence from the GFP was not detected in these assays, and the FACS profile was similar to untreated controls. The flow cytometry data for each experiment was representative of at least three replicates. (C) Anti-vWf staining was detected with a FITC-labeled secondary antibody used to identify endothelial cells by flow cytometry, and this was compared to parallel collagenase-dissociated untreated CAM cells incubated without primary antibody. (D) Immunolocalization of endogenous von Willebrand factor in collagenase-dissociated untreated permeabilized CAM cells (arrowhead) replated on 3 μg/ml collagen and detected with a fluorescent secondary antibody (bar, 10 μm). Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)

Figure 4 Retroviral Delivery of Src 251 and Csk in a Subcutaneous Murine Angiogenesis Model (A) Angiogenesis was induced by a subcutaneous injection of growth factor-depleted Matrigel containing saline or VEGF (400 ng/ml) with 2 × 106 ecotropic packaging cells expressing GFP retrovirus in the flank of athymic wehi (nu/nu) mice and analyzed after 5 days of incubation. The neovascularization was quantitated by immunoblotting with a VEGF receptor antibody (flk-1) that is specific for endothelial cells. (B) The effects of kinase-deleted Src 251, Csk, or GFP retrovirus on VEGF- (400 ng/ml) or bFGF- (400 ng/ml) induced angiogenesis was analyzed by immunoblotting the tissue lysates with an anti-flk-1 antibody. (C) The effect of the Src 251– and Csk-expressing retroviruses on VEGF-induced neovascularization was quantified by enumerating the number of CD34 positive vessels in tissue cross sections by indirect immunofluorescence in triplicate random fields at 20× as described in the Experimental Procedures. Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)

Figure 5 The Effect of VEGF-Induced Vascular Leakage in the Ears and Brains of src−/− and src+/− Mice (A) Gene delivery of the human VEGF cDNA in an adenovirus vector was injected intradermally in the right ear of src+/− or src−/− mice, and the neovascularization of the ears were photographed after 5 days of expression. Adenovirus expressing β-galactosidase was injected into the left ears as a negative control. Staining for β-galactosidase in these ears confirmed similar adenovirus expression in each genetic background. Scale bar, 1 mm; n = 4. (B) VEGF or saline was stereotactically injected into the left or right frontal lobes, respectively, of src+/− or src−/−. After injection with Evan's blue and perfusion, the brains were removed and photographed with a stereoscope (6×, final magnification; arrowhead, injection site). (C) Cross sections of the above VEGF- or saline-injected brains from src+/− or src−/− mice were prepared and analyzed for VEGF-induced VP by confocal microscopy to visualize the fluorescence of the extravasated Evan's blue (6×, final magnification; arrowhead, injection site). Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)

Figure 6 Miles Assay for Vascular Permeability of VEGF in the Skin of Mice Deficient in Src, Fyn, or Yes (A) The vascular permeability properties of VEGF in the skin of src+/− (upper) or src−/− (lower) mice was determined by intradermal injection of saline or VEGF (400 ng) into mice that have been intravenously injected with Evan's blue dye. After 15 min, skin patches were photographed (scale bar, 1 mm). Arrowheads indicate the injection sites. (B) The regions surrounding the injection sites of the VEGF, bFGF, or saline were dissected, and the permeability quantitated by elution of the Evan's blue in formamide at 56°C for 24 hr, and the absorbance measured at 600 nm (left). The ability of an inflammation mediator (allyl isothiocyanate), known to induce inflammation-related VP, was tested in src+/− or src−/− mice (right). (C) The ability of VEGF to induce VP was compared in src−/−, fyn−/−, or yes−/− mice in the Miles assay. Data for each of the Miles assays are expressed as the mean ± SD of triplicate animals. src−/− and yes−/− VP defects compared to control animals were statistically significant (*p < 0.05, paired t test), whereas the VP defects in neither the VEGF-treated fyn−/− mice nor the allyl isothiocyanate–treated src−/− mice were statistically significant (**p < 0.05). Molecular Cell 1999 4, 915-924DOI: (10.1016/S1097-2765(00)80221-X)