Disruption of endothelial FGF signaling affects vessel integrity and permeability.

The tracheal vasculature is known to be disrupted by a systemic expression of circulating VEGF traps (1). Following systemic expression of sFGFR1IIIc, we observed a loss of vascular integrity in the tracheal circulation as demonstrated by extravasation of 100 nm i.v.-injected microsphere particles that was not present in mice exposed to the control adenovirus (Figure ​(Figure2A).2A). To further characterize this loss of vascular integrity, we evaluated vascular permeability in normal C57BL/6 mice following systemic expression of either sFGFR1IIIc, sFGFR3IIIb, or a control construct. In agreement with the observed loss of tracheal vasculature integrity, there was a substantial increase in Evans blue dye retention in the skeletal muscle in mice exposed to sFGFR1IIIc but not sFGFR3IIIb virus and control mice (Figure ​(Figure2B).2B). The same Evans blue assay demonstrated a significant increase in permeability in both myocardial and pulmonary tissue in sFGFR1IIIc- and sFGFR3IIIc-exposed but not control or sFGFR3IIIb mice (Figure ​(Figure2C). 2C).

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Figure 2

Impaired vascular integrity in mice lacking FGF signaling.

(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.

To assess the effect of prolonged suppression of FGF signaling, nude mice were injected with Ad-FGFR1IIIc-Fc. After 8 weeks of exposure of sFGFR1IIIc trap, the mortality rate was 15.3%, unlike C57BL/6 mice, which showed very little mortality. Histological examination showed disrupted vascular integrity in the tracheal circulation (Figure ​(Figure2D)2D) and scattered areas of arterial thrombosis and hemorrhage in the lungs and heart (Figure ​(Figure2E). 2E).

To obtain an insight into the cause of this loss of vascular integrity, arterial endothelium of a normal rat femoral artery was exposed in vivo to either the Ad-FGFR1DN or Ad-Null virus. FGFR1 dominant negative construct (FGFR1DN), a cytoplasmic truncated form of mouse FGFR1IIIc, is capable of suppressing signaling of all 4 FGFRs by heterodimerizing with other FGFR isoforms (16, 21).

Two days after viral transduction, enface VE-cadherin staining demonstrated normal endothelial morphology in the artery exposed to Ad-Null, while there was a loss of endothelial cell-cell contacts in the artery exposed to FGFR1DN (Figure ​(Figure3A).3A). Five days later, the majority of endothelial cells in the Ad-FGFR1DN–exposed artery appeared round and not in contact with each other, indicating a further loss of cell-cell contact (Figure ​(Figure3A).3A). Remarkably, endothelial cells were no longer aligned along the axis of the blood flow, suggesting the loss of either the ability to sense the shear stress or to respond to it. Similar findings, albeit somewhat less dramatic, were observed both in the arterial and venous systems of mice with systemic expression of the sFGFR1IIIc-Fc trap, suggesting that the FGF inhibition effect is not restricted to the arterial vasculature (Figure ​(Figure3B). 3B).

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Figure 3

In vivo effect of FGF inhibition in the endothelium.

(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.

Scanning electron microscopy (SEM) confirmed the loss of cell-cell contacts (Figure ​(Figure4B).4B). Furthermore, several areas of the arterial segment exposed to FGFR1DN demonstrated a complete loss of endothelial lining, leading to the exposure of the underlying basement membrane and platelet adhesion (Figure ​(Figure4B).4B). To further evaluate the appearance of endothelial junctions, we employed transmission electron microscopy. Whereas endothelial junctions exposed with control virus were tightly sealed (Figure ​(Figure4C),4C), FGFR1DN-transduced endothelium demonstrated open junctions and detachment of endothelial cells (Figure ​(Figure4,4, D–F)

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Figure 4

Effect of FGF inhibition in the endothelium: electron microscopy analyses.

(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.

To further examine the effect of suppression of endothelial FGF signaling on permeability, we carried out in vitro studies using both FGFR1DN and sFGFRs. Ad-FGFR1DN transduction of BAEC or exposure of these cells to sFGFR1IIIc or sFGFR3IIIc trap resulted in a significant decrease in transendothelial impedance compared with control cells 48 hours later (Figure ​(Figure5,5, A and B). Furthermore, the Ad-FGFR1DN–transduced monolayer was permeable to 70 kDa and 2000 kDa (2 MDa) dextran, suggesting the presence of large gaps in the monolayer (Figure ​(Figure5C).5C). Exposure of the mature BAEC monolayer to sFGFR1IIIc but not FGFR3IIIb traps also significantly increased permeability to 70 kDa and 2 MDa dextran (Figure ​(Figure5D). 5D).

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Figure 5

Increased endothelial permeability in cells lacking FGF signaling.

(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.

FGF signaling is required for maintenance of adherens junctions.

The 2 types of junctions found in the endothelium and contributing to structural stability are adherens and tight junctions (22). Many reports support the idea that tight junction organization is largely dependent on the formation of adherens junctions. In some cellular systems, blocking adherens junctions inhibits the correct organization of tight junctions (23). Adherens junctions play a critical role not only in regulation of vascular permeability but also in maintenance of vessel stability (24). Since VE-cadherin is the principal protein regulating formation of adherens junctions in endothelial cells, we evaluated its expression in Ad-FGFR1DN–transduced endothelial cells in vitro. BAEC monolayers were transduced when fully confluent at an MOI ensuring approximately 30%–50% transduction to avoid possible toxic adenoviral effects. In cells expressing the dominant negative construct, regular VE-cadherin staining at adherens junctions at the cell periphery showed interruptions that correlated with gaps in the monolayer by phase contrast or differential interference contrast. No such changes were observed in Ad-GFP–transduced cells (Figure ​(Figure6A).6A). Exposure to the Ad-FGFR1IIIc-Fc–encoded trap led to a similar loss of junctional VE-cadherin expression (Figure ​(Figure6B). 6B).

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Figure 6

Junction proteins are absent from cell-cell contacts in cells lacking FGF signaling.

(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.

The same cells also demonstrated a loss of tight junctions as demonstrated by zonula occludens-1 (ZO-1) staining (Figure ​(Figure7A).7A). The effect of suppression of FGF signaling on cell membrane VE-cadherin expression was not limited to the arterial endothelium, as transduction of human saphenous vein endothelial cells (HSVECs) with Ad-FGFR1DN demonstrated a similar loss of VE-cadherin from cell-cell junctions (Figure ​(Figure7B).7B). Furthermore, quantitative analysis of FGFR1DN-transduced cells confirmed visual observations. The number of cells with interrupted junctional VE-cadherin staining increased as the viral dose of Ad-FGFR1DN but not Ad-GFP increased, while overall cell density remained unaltered, suggesting that the effect of FGF signaling disruption on VE-cadherin expression is not due to cell loss (Supplemental Figure 1, A and B; supplemental material available online with this article; 10.1172/JCI35298DS1). Moreover, Western blotting demonstrated no increase in caspase 3 cleavage in FGFR1DN-expressing endothelial cells compared with controls (Supplemental Figure 1D).

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Figure 7

Tight junction and venous cells are similarly affected by FGF signaling inhibition.

(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.

In order to examine these cells more closely, we employed SEM to examine Ad-GFP– and Ad-FGFR1DN–transduced endothelial monolayers in vitro. Ad-GFP–expressing monolayer appeared continuous with rare gaps that normally occur in endothelial cells in culture (Supplemental Figure 1C). Conversely, Ad-FGFR1DN–expressing monolayers demonstrated a sharp increase in the number and size of gaps within the monolayer (Supplemental Figure 1C) as soon as 24 hours after transduction that was then followed by a progressive disassembly of the monolayer. Since the data presented above suggested that FGFs serve to protect vascular integrity, we measured endothelial cell monolayer permeability in vitro after FGF and VEGF exposure. In agreement with previous results (25), exposure to VEGF-A markedly decreased monolayer impedance. In contrast, exposure to FGF1 slightly but significantly increased impedance (and thus reduced permeability) (control, 1652.68 ± 58.3 ohms; FGF1: 1776.08 ± 48.62 ohms at 8 hours, P < 0.05). Furthermore, pretreatment with low concentration of FGF prevented VEGF-induced increase of monolayer permeability (Figure ​(Figure8A).8A). Moreover, while under conditions in which a confluent endothelial monolayer was maintained without serum supplementation, VEGF-A rapidly disassembled adherens junctions as demonstrated by movement of VE-cadherin from cell-cell contacts into the cytosol (Figure ​(Figure8B).8B). However, such an effect was not observed with FGF1 treatment (Figure ​(Figure8B).8B). Thus, these experiments demonstrate that FGF signaling regulates endothelial permeability and vascular integrity in vitro and in vivo.

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Figure 8

FGF treatment maintains EC junctions and decreases endothelial permeability in vitro.

(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.

Antibodies.

Antibodies used for immunoprecipitation, Western blotting, and immunofluorescence include anti–phospho- and total Erk1/2, anti–β-catenin, and anti–caspase-3 (Cell Signaling Technology), anti–VE-cadherin (Santa Cruz Biotechnology Inc. and Research Diagnostics Inc.), anti–p120-catenin (BD Biosciences — BD Transduction Laboratories), anti–ZO-1 (Invitrogen), N-cadherin (Zymed Laboratories Inc.), and anti–β-tubulin (Sigma-Aldrich).

Cell culture and adenoviral transduction.

BAECs and HSVECs were cultured at 37°C in 5% CO₂ in EGM-2 MV medium (Cambrex) or MCDB-131 complete medium (VEC Technologies Inc.), respectively, and precoated with fibronectin (10 μg/ml) unless otherwise specified. Adenoviral vectors were transduced at an MOI of 10–100 PFUs/cell for Ad-FGFR1DN or 50–200 viral particles/cell for Ad-sFGFRs. The infection medium was replaced 4–6 hours later with the normal growth medium.

Western blotting and immunoprecipitation.

The procedure described previously was applied with small modifications (51). In brief, cells were washed twice with ice-cold Ca²⁺ and Mg²⁺ PBS and lysed in RIPA buffer: 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM CaCl₂, 1× complete protease inhibitor mixture (Roche), and 1 mM sodium orthovanadate. For Erk1/2 phosphorylation studies, to obviate possible quenching of signal that occurs when probing the same protein using different antibodies on the same membrane, the identical samples were run in duplicate and membranes were blotted with either phospho-Erk1/2 or total-Erk antibody.

For immunoprecipitation, samples were precleared on protein A/G beads (Calbiochem) for 1 hour at 4°C. VE-cadherin was immunoprecipitated from 500 μg of extract using 2 μg of anti–VE-cadherin antibody followed by 20 μl of protein A/G beads. Beads were washed 5 times using cold PBS, boiled in SDS-PAGE loading buffer, and resolved by SDS-PAGE. Where indicated, cells were treated prior to lysis with 100 μM PV for 5 minutes at 37°C to inhibit tyrosine phosphatases.

Immunofluorescence.

Immunocytochemistry was performed with a standard procedure using antibodies described above. In brief, cells were fixed with 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. Incubation with the primary antibody was followed by incubation with Alexa Fluor–conjugated secondary antibodies (Invitrogen). Samples were examined under a Zeiss LSM 510 confocal microscope.

Biotinylation of cell surface protein.

Twenty-four hours after adenovirus transduction, cells were washed twice with ice-cold PBS and incubated with 0.5 mg/ml sulfo-N-hydroxysuccinimide ester long-chain biotin (Pierce Biotechnology) in PBS, pH 7.4, for 15 minutes at 4°C. The biotinylation reaction was quenched with ice-cold 1% BSA-PBS. Cell lysate was incubated with immobilized NeutrAvidin (Pierce Biotechnology) at 4°C to separate biotinylated proteins.

Ad-sFGFR infection and measurement of sFGFR in mouse blood and tissue.

All animal experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee of Dartmouth College.

Wild-type male C57BL/6 mice or male NU/NU mice (Charles River laboratories) were injected with 5 × 10¹⁰ viral particles from the tail vein in conjunction with 10 μM antennapedia peptide (52) (gift from W.C. Sessa, Yale University School of Medicine, New Haven, Connecticut, USA). Plasma and tissue levels of sFGFRs were measured using a human IgG ELISA quantitation kit (Bethyl Laboratories Inc.).

Permeability assays.

0.5% Evans blue dye (200 μl) was injected into the retroorbital space and was allowed to circulate for 30 minutes. The mice were then sacrificed, blood was drained by perfusion with saline, and the gastrocnemius muscle, lungs, and heart were excised and dried at 55°C. Evans blue dye in tissues was extracted with formamide for 24 hours at 55°C and its fluorescence at 607 nm was measured by a fluorescent reader (BioTek).

Transwell tracer experiments with fluorescent dextran were performed using the HTS Fluoroblok 24-well Transwell system (1.0 μm pore; BD). BAECs (2 × 10⁴) were plated on a Transwell insert membrane coated with 10 μg/ml fibronectin. The monolayer was matured until full confluence and transduced with adenoviruses. Thereafter, 2 MDa FITC-dextran was added in the upper chamber and the fluorescence of the lower chamber was measured at fixed time points.

Transendothelial electrical resistance, an index of endothelial cell barrier function, was measured in real time using an electric cell-substrate impedance sensing (ECIS) system (Applied BioPhysics) (53). BAECs (5 × 10⁴ per well) were plated on sterile 8-chambered gold-plated electrode arrays (8W10E) precoated with fibronectin (10 μg/ml) and grown to full confluence. Immediately after adenoviral transduction, the electrode arrays were mounted on the ECIS system within an incubator (37°C, 5% CO₂) and connected to its recorder device. Monolayer resistance was recorded for 50 hours in 5-minute intervals. After 6-hour exposure to adenovirus, viral-containing medium was replaced with normal growth medium (EGM-2 MV).

Adenoviral transduction and enface VE-cadherin staining of artery.

Adenovirus (10⁹ PFUs) premixed with the 10 μM antennapedia peptide (52) was transduced in the rat femoral artery as described previously (54). The artery was excised and cut open longitudinally to expose the endothelium. The arterial segment was placed with the endothelial side up and fixed with tungsten pins (Fine Science Tools). After fixation with 2% paraformaldehyde in PBS, standard immunostaining was performed using anti–VE-cadherin (Santa Cruz Biotechnology Inc.) and Alexa Fluor 568–conjugated anti-goat antibody (Invitrogen). Using a Zeiss LSM 510 confocal microscope, 20–30 Z-Stack 1-μm optical sections were acquired and projected to a single image plane. To examine the effect of sFGFR, wild-type mice were infected (5 × 10¹⁰ viral particles with 10 μM antennapedia) and the carotid artery and jugular vein were stained for VE-cadherin and visualized as described above.

Mouse trachea vasculature studies.

Sites of leakage in mouse tracheal blood vessels were visualized using 100 nm extravasated green fluorescent polymer microspheres (Duke Scientific Corp.) as described previously (55). In brief, 10 days after injection of control or sFGFR1IIIc adenovirus, anesthetized mice received 20 μl (4 × 10¹¹ particles) i.v. injection of fluorescent microspheres. Two minutes later, intravascular microspheres were removed from the bloodstream by perfusion of 1% paraformaldehyde in PBS for 2 minutes at a pressure of 120 mmHg. Tracheas were removed from mice and immersion fixed for an additional 2 hours, and then the vasculature was incubated with anti-CD31 antibody (Chemicon) and subsequently with Cy3-conjugated goat anti-hamster IgG (Jackson ImmunoResearch). Trachea whole mounts were examined using a Zeiss LSM 510 confocal microscope.

Electron microscopy: cells.

BAECs were grown to confluence on fibronectin-coated 12-mm glass coverslips at which point they were transduced with either Ad-GFP or Ad-FGFR1DN-GFP as described. After 24 hours, the cells were fixed (30 minutes, room temperature [RT]) in 2% glutaraldehyde in 0.1 M sodium cacodylate, rinsed (2 × 10 minutes, RT) in 0.1 M sodium cacodylate, and postfixed (1 hour, RT, in dark) in 1% OsO₄ in 0.1 M sodium cacodylate. The cells were dehydrated in graded ethanols, critical point dried using a Samdri-795 Critical Point Dryer (Tousimis), coated with a 3-nm amorphous osmium layer in an OPC-60 Osmium Plasma Coater (Filgen), mounted on stubs, and examined at high voltage in an XL-30 ESEM-FEG microscope (FEI Co.) using secondary electron detection.

Electron microscopy: artery specimens.

The right femoral artery of male rats was used for epigenetic expression via local adenoviral delivery, as described above. The femoral artery segments that were treated with either Ad-Null or Ad-FGFR1DN as well as the contralateral control segments were removed, rinsed in PBS, and cut open longitudinally. The artery fragments were opened flat with the endothelium facing upwards, pinned down using fine tungsten pins, and fixed (1 hour, RT) in 2% glutaraldehyde. At all times during these procedures, the tissue was submerged in liquid. The arteries were then processed for electron microscopy as described for the cells.

This work has been supported by NIH grants HL-53793 (to M. Simons) and HL-65418 (to R.V. Stan). We would like to thank Amy Hall and Samantha Shipman (Dartmouth Medical School) for technical assistance and mouse handling and Kenneth Orndorff at the Englert Cell Analysis Laboratory (Dartmouth Medical School) for technical assistance for the confocal microscope system. We would like to thank Frank Giordano and Reed Hickey (Yale University School of Medicine) for adenovirus generation.