(A) Enface preparation of the rat femoral artery transduced with Ad-Null or Ad-FGFR1DN (10⁹ PFUs). Segments of arteries were transduced with adenoviruses and stained for VE-cadherin (red) and maximum intensity projection of 1-μm Z-Stack sections is shown. At day 5, endothelial cells lost cell-cell contacts and gaps formed between cells. Scale bars: 10 μm. (B) Mouse carotid artery and jugular vein exposed to systemic Ad-Null or Ad-sFGFR1IIIc viruses. Segments of arteries were stained for VE-cadherin, and maximum intensity projection of 1-μm Z-Stack sections is shown. Scale bars: 20 μm.

(A) Immunostaining of quiescent endothelial monolayer (BAECs) transduced with Ad-GFP or Ad-FGFR1DN-GFP. Cells were stained for VE-cadherin (red) and ZO-1 (green). The Ad-FGFR1DN-GFP vector has a bidirectional promoter encoding both GFP and FGFR1DN as described above (GFP signal is shown in white). Arrows indicate reduced VE-cadherin and ZO-1 staining and gaps between endothelial cells. Scale bars: 20 μm. (B) Immunostaining of quiescent venous endothelial monolayer. HSVECs were transduced with Ad-GFP or Ad-FGFR1DN-GFP and stained for VE-cadherin (red) and p120-catenin (green) 24 hours later. Note loss of VE-cadherin and p120-catenin staining at cell-cell contacts and formation of intercellular gaps of FGFR1DN-GFP–transduced cells (white arrows). Scale bars: 20 μm.

(A) Immunostaining of quiescent and fully confluent endothelial monolayer. BAECs were transduced with Ad-GFP or Ad-FGFR1DN-GFP using low MOI (10 and 25, respectively) to limit the number of transduced cells and minimize virus-mediated toxicity. Cells were stained for VE-cadherin (red) and β-catenin (green) 24 hours later. The Ad-FGFR1DN-GFP vector has a bidirectional promoter encoding both GFP and FGFR1DN, thus marking transduced cells with GFP (shown in white). Note the loss of VE-cadherin and β-catenin staining at cell-cell contacts of FGFR1DN-GFP–transduced cells. Arrow points to the gap between neighboring FGFR1DN-GFP–transduced cells. Scale bars: 20 μm. (B) Immunostaining of quiescent endothelial monolayer (BAECs) transduced with Ad-Null or Ad-sFGFR1IIIc. Cells were stained for VE-cadherin (red), p120-catenin (green), and DAPI (blue). sFGFR was secreted in the medium; the effect is not limited to the transduced cells. Arrows indicate gaps between endothelial cells. Scale bars: 20 μm.

(A) ECIS permeability assay using confluent monolayer. BAECs are matured to full confluency and the medium was replaced to 1% BSA in EBM-2 (Cambrex). FGF1 (1 ng/ml) or 1% BSA/EBM-2 was added at the indicated time point (arrow). Six hours later, VEGF-A (25 ng/ml) or 1% BSA/EBM2 was added at the indicated time point (arrow). At 6 hours, FGF treatment significantly increased monolayer impedance compared with the control treatment. (B) FGF treatment does not induce junction disruption on endothelial cell monolayers. BAECs are matured to full confluency, starved with 1% BSA in EBM-2 for 48 hours, and treated with either FGF1 (50 ng/ml) or VEGF (80 ng/ml) for 30 minutes Note with this condition, VEGF rapidly disrupts endothelial cell junctions (white arrows) whereas FGF treatment maintains junctions similar to those in the control monolayer. Scale bars: 20 μm.

(A) Increased vascular permeability and impaired endothelial morphology in sFGFR1IIIc mouse. Ten days after adenoviral injection in C57BL/6 mice, the trachea vasculature was stained for CD31 (red). Green represents microspheres in the extravascular space. Scale bars: 20 μm (left panels); 10 μm (right panels). (B) sFGFR1IIIc increases vascular permeability in the muscle. Ad-Null (control) or Ad-sFGFR was injected in C57BL/6 mice, and 14 days later, Evans blue dye was injected i.v. The adductor group muscles were harvested for quantitation. Data are shown as mean ± SD (n = 4). *P < 0.05, control vs. sFGFR1IIIc by t test. (C) Inhibition of FGF signaling in the heart and lung increases vascular permeability. Ad-Null (control), Ad-sFGFR was injected in C57BL/6 mice, and 10 days later, Evans blue dye levels were examined in the heart and lung. Data are shown as mean ± SD for n = 7, control; n = 8, sFGFR1IIIc; n = 4, sFGFR3IIIb; n = 4, sFGFR3IIIc. *P < 0.05, vs. control by t test. (D) Increased vascular permeability in Ad-sFGFR1IIIc–treated nude mouse. NU/NU nude mice (Charles River Laboratories) were subjected to trachea CD31. Junctional CD31 staining observed in control (arrows) is completely absent in the Ad-sFGFR1IIIc–treated trachea vasculature. Scale bars: 20 μm (left panels); 10 μm (right panels). (E) Hemorrhage observed in Ad-sFGFR1IIIc–treated mouse lung and heart. Ten days after adenoviral injection in nude mice, lung and heart were harvested for H&E staining. Upper panels show lung sections and lower panels show heart sections. Black arrows indicate myocardial hemorrhage. Original magnification, ×400.

(A) Endothelial monolayer permeability evaluated with ECIS system. Impedance was measured every 5 minutes for 48 hours after the onset of adenoviral transduction. After adenoviral exposure, cell culture medium was changed to normal growth medium. n = 3 in each group, P < 0.05, control versus FGFR1DN or sFGFR1IIIc at 48 hours (B) ESIS analysis using various sFGFR adenoviruses. After adenoviral exposure, medium was replaced with normal growth medium. n = 3 in each group, P < 0.05, control versus sFGFR1IIIc or sFGFR3IIIc at 48 hours (C) Transwell tracer experiment using high (2 MDa) and low (70 kDa) molecular weight dextran. Full confluent BAECs on the Transwell chambers were transduced and dextrans were added in the upper chamber. Fluorescent values in the lower chamber were measured at indicated time points using a fluorescent microplate reader. Three independent experiments were performed. One representative experiment is shown as mean ± SD for n = 3 in each group; *P < 0.05 by Student’s t test, control vs. FGFR1DN. AU represents relative intensity of fluorescence. (D) Transwell tracer experiment using Ad-sFGFRs. Data are shown as mean ± SD for n = 3 in each group; *P < 0.05 by Student’s t test, control vs. sFGFR1IIIc.

(A) Decreased cell surface VE-cadherin in Ad-FGFR1DN–transduced BAECs. Quiescent BAECs were transduced with Ad-GFP or Ad-FGFR1DN and the monolayer was treated with biotin to label cell surface proteins. Biotinylated proteins were subjected to Western blot analysis and probed for VE-cadherin or N-cadherin. Total cell lysates from the same samples were also analyzed by Western blotting to evaluate total protein expression. (B) Western blot analysis of expression of junction proteins. Transduced BAECs were harvested at the indicated time points, and total cell lysates were analyzed by Western blotting. (C) Effect of FGF treatment on VE-cadherin interaction with catenins. BAEC monolayers were maintained in 0.5% FBS basal medium for 24 hours and then stimulated with FGF1 (50 ng/ml) for the indicated times. Total cell lysates were immunoprecipitated with anti–VE-cadherin antibody and blotted with p120-catenin, β-catenin, or VE-cadherin antibody. (D) FGFR signaling requirement for VE-cadherin interaction with catenins. Subconfluent BAEC monolayer (~70% confluency) was transduced with Ad-GFP or Ad-FGFR1DN, and cells were grown to form mature junctions. At indicated time points, total cell lysates were prepared and immunoprecipitated with VE-cadherin antibody followed by immunoblotting with p120-catenin, β-catenin, or VE-cadherin antibody. As the monolayer forms mature junctions, VE-cadherin/p120-catenin interaction increases (Ad-GFP), whereas this interaction was decreased in Ad-FGFR1DN–treated cells. Nontransduced cells were treated with 100 μM PV for 10 minutes prior to cell lysis, showing disrupted VE-cadherin/p120 interaction.

(A) sFGFR blocks FGF-induced Erk1/2 phosphorylation in vitro in a dose-dependent manner. Ad-GFP and Ad-sFGFR1IIIc were transduced in BAECs and stimulated with indicated concentration of FGF1 for 10 minutes. Total cell lysates were subjected to Western blotting. p-Erk1/2, phospho-Erk1/2; t-Erk1/2, total Erk1/2; VP, viral particles. (B) sFGFRs effectively inhibit FGF1-induced Erk1/2 activation. Ad-Null, Ad-sFGFR1IIIc, Ad-sFGFR3IIIb, and Ad-sFGFR3IIIc were transduced in BAECs and stimulated with FGF1 for 5 minutes. (C) sFGFR3IIIb does not inhibit FGF2-induced Erk1/2 activation. Ad-Null and Ad-sFGFRs were transduced in BAECs and stimulated with FGF2 for 5 minutes. (D) Plasma expression levels of sFGFR in mice. Ad-Null (control) or Ad-sFGFR1IIIc (5 × 10¹⁰ viral particles) was injected into C57BL/6 mice, and blood samples were taken at indicated time points. sFGFR levels were measured using an ELISA system detecting human IgG-Fc. Data are shown as mean ± SD, n = 4 in each group. (E) Tissue distribution of sFGFR. After 10 days of Ad-Null (control) or Ad-sFGFR1IIIc injection (5 × 10¹⁰ viral particles), tissue samples were collected and total protein was extracted. Thereafter, ELISA assays detecting human IgG-Fc were performed. Data are shown as mean ± SD, 4 animals in each group. (F) Body weight change after injection of sFGFR. Ten-week-old C57BL/6 mice were injected with adenoviruses (5 × 10¹⁰ viral particles), and body weight was measured weekly. Data are shown as mean ± SD, n = 4 in each group. *P < 0.05, Student’s t test.

(A) SEM analysis of rat femoral arteries transduced with Ad-Null. The arterial wall shows ridges formed due to contractions in the underlying smooth muscle cells (top panel, scale bar: 50 μm). Higher magnification detail of the Ad-Null–infected artery demonstrates the continuous endothelial monolayer (bottom panel, scale bar: 10 μm). (B) SEM analysis of Ad-FGFR1DN–transduced rat femoral artery. The area with remaining endothelial cells is shown (left panels, scale bars: 50 μm [top]; 10 μm [bottom]). Endothelial cells are swollen and have lost contacts with neighboring cells. Underlying matrix layer is partially exposed (white arrows). Area demonstrating severe loss of the endothelium with exposure of the subjacent basement membrane is also shown (right panels, scale bar: 50 μm [top]). Higher magnification shows a few cells, presumably platelets by size, adhering to the vessel wall (white arrowheads) (right panels, scale bar: 10 μm [bottom]). (C) Transmission electron microscopy analysis of Ad-Null–transduced rat femoral artery (day 2). Adjacent endothelial cells form a tightly sealed junction indicated by black arrows. Scale bar: 500 nm. (D) Ad-FGFR1DN–transduced artery (day 1). Scale bar: 2 μm. Inset: a gap is formed between endothelial cells. Arrow (inset) indicates open cell-cell junction. Scale bar: 500 nm. (E) Ad-FGFR1DN–transduced artery (day 2) shows an open endothelial junction (black arrows). A platelet (PLT) adheres to the open space and extends a cellular process (white arrowhead). Scale bar: 500 nm. (F) Ad-FGFR1DN–transduced artery showing a wide gap in the endothelium (black arrow) and an EC detaching from the basement membrane. Scale bar: 2 μm.