Integrin ?v?5 Regulates Lung Vascular Permeability
and Pulmonary Endothelial Barrier Function
George Su, Maki Hodnett, Nanyan Wu, Amha Atakilit, Cynthia Kosinski, Mika Godzich, Xiao Zhu Huang,
Jiyeun K. Kim, James A. Frank, Michael A. Matthay, Dean Sheppard*, and Jean-Franc ¸ois Pittet*
Lung Biology Center, Division of Pulmonary and Critical Care Medicine, Laboratory of Surgical Research, Departments of Anesthesia and
Surgery, and Cardiovascular Research Institute, University of California, San Francisco; and Veterans Administration Medical Center,
San Francisco, California
Increased lung vascular permeability is an important contributor
to respiratory failure in acute lung injury (ALI). We found that a
function-blocking antibody against the integrin ?v?5 prevented
development of lung vascular permeability in two different models
of ALI: ischemia-reperfusion in rats (mediated by vascular endothe-
lial growth factor [VEGF]) and ventilation-induced lung injury (VILI)
in mice (mediated, at least inpart, by transforming growth factor-?
[TGF-?]). Knockout mice homozygous for a null mutation of the
integrin ?5 subunit were also protected from lung vascular perme-
ability in VILI. In pulmonary endothelial cells, both the genetic
absence and blocking of ?v?5 prevented increases in monolayer
permeability induced by VEGF, TGF-?, and thrombin. Furthermore,
actin stress fiber formation induced by each of these agonists was
attenuated by blocking ?v?5, suggesting that ?v?5 regulates in-
duced pulmonary endothelial permeability by facilitating interac-
tions with the actin cytoskeleton. These results identify integrin
?v?5 as a central regulator of increased pulmonary vascular perme-
ability and a potentially attractive therapeutic target in ALI.
Keywords: integrin ?v?5; lung vascular permeability; pulmonary
endothelial barrier function
Acute lung injury (ALI) is a devastating clinical syndrome char-
acterized by development of pulmonary edema and flooding
of alveolar spaces leading to impaired gas exchange, arterial
hypoxemia, and respiratory failure (1). While much progress
has been made in understanding the pathogenesis of ALI, it is
estimated that 190,600 cases of ALI occur every year in the
United States alone; these are associated with 74,500 deaths and
3.6 million hospital days (2). Effective pharmacologic therapies
are not currently available and the molecular mechanisms regu-
lating ALI remain poorly understood.
Vascular permeability in the lung has long been considered
a principalpathologic hallmark ofALIthat is largely responsible
for its characteristic pulmonary edema formation (3, 4). Re-
cently, integrin ?v?5, a member of the integrin family of hetero-
dimeric transmembrane cell surface receptors, was shown to
specifically regulate increases in vascular permeability induced
by vascular endothelial growth factor (VEGF) in the systemic
circulation (5). Although regulation of permeability in the sys-
temic and pulmonary circulations is often physiologically dis-
(Received in original form July 4, 2006 and in final form September 21, 2006)
* These authors contributed equally to this manuscript.
HL66600 (Baygenomics) from the NHLBI (to D.S.), HL074005 (SCCOR, Project 4)
(to J.-F.P.), and UCTRDRP 12FT-0123 (to G.S.)
Correspondence and requests for reprints should be addressedto Dean Sheppard,
Lung Biology Center, UCSF, Rock Hall, Room 545, 1550 4th Street, San Francisco,
CA 94158. E-mail: firstname.lastname@example.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2006-0238OC on November 1, 2006
Internet address: www.atsjournals.org
Vol 36. pp 377–386, 2007
We describe a novel role for integrin ?v?5 in regulating
lung vascular permeability and agonist-induced endothelial
of the actin-cytoskeleton may be a mechanism responsible
for these effects.
we hypothesized that ?v?5 could be an important regulator
of vascular permeability in the lung. Therefore, we sought to
ity in in vivo models of ALI.
In this report, we used two in vivo models of ALI to examine
the role of ?v?5 inregulating lungvascular permeability:ischemia-
reperfusion (IR)-induced and ventilator-induced lung injury
(VILI). IR-induced lung injury is a significant clinical problem
in cardiac surgery and, in particular, with lung transplantation
(6). Although the pulmonary edema associated with lung trans-
uted to IR can occur in up to 20% of patients, leading to pro-
longed post-transplant length of hospitalization and increased
post-transplant mortality (7). Mechanical ventilation, while con-
sidered an essential tool for managing patients with respiratory
failure, is now itself recognized, when administered at high tidal
volumes, as an important contributing factor to the development
of pulmonary edema (VILI) (1, 8, 9).
Our studies show that ?v?5 regulates lung vascular perme-
ability in models of both IR and VILI. However, in the lung,
as opposed to what has been described in the systemic vascula-
to VEGF-induced effects alone; in pulmonary vascular endothe-
lial cells, both genetic absence and blockade of ?v?5 prevented
monolayer permeability induced by three very different ede-
magenicagonists—VEGF,TGF-?, andthrombin.Previous stud-
ieshaveidentified the inductionofactinstressfibers asan impor-
tant step in regulating agonist-induced increases in endothelial
by all three agonists was attenuated by blockade of ?v?5, sug-
gesting a mechanism for how ?v?5 might regulate paracellular
endothelial permeability in the lung downstream of multiple
signaling pathways. Understanding how ?v?5 regulates pulmo-
nary endothelial permeability could provide valuable insights
identify this integrin as a promising target for the treatment of
MATERIALS AND METHODS
Reagents and Antibodies
VEGF (R&D Systems, Minneapolis, MN), TGF-? (R&D Systems),
thrombin (Amersham Biosciences, Piscataway, NJ), RhoA kinase
378AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 362007
(ROCK) inhibitor (Y-27632) (Calbiochem, San Diego, CA), VEGF
receptor II-Ig chimera adenovirus (AdVEGFRII-Ig) (generous gift from
Richard C. Mulligan, Harvard School of Medicine , Boston, MA),
green fluorescent protein (GFP) adenovirus control (AdGFP) (gener-
ous gift from George Davis, Texas A&M University, College Station,
TX), anti-integrin ?v?6 antibody (3G9) and TGF-? type II receptor
IgG chimera (TGF-?-RII-Ig) (generous gifts from Paul Weinreb, Bio-
gen Idec,Cambridge, MA),IgG2b isotype antibody: mouse anti-human
low-density receptor (LDL) receptor antibody (CRL-1691, clone C7;
antibody (LM609) (Chemicon, Temecula, CA), anti-integrin ?v?8 anti-
body (37E.1) (generous gift from Steve Nishimura, University of Cali-
fornia, San Francisco, CA), collagenase A (Sigma, St. Louis, MO),
heparin (Sigma), M-450 Dynabeads (Dynal, Carlsbad, CA), anti-Fc?
receptor II/III antibody (BD Pharmingen, San Jose, CA), anti-intracel-
lular adhesion molecule (ICAM)-2 antibody (BD Pharmingen and
Santa Cruz Biotechnology, Santa Cruz, CA), platelet endothelial cell
adhesion molecule (PECAM, CD-31) (BioLegend, San Diego, CA),
anti-vascular endothelial (VE)-cadherin antibody (Santa Cruz Biotech-
nology), anti-CD34 antibody (BD Pharmingen),
albumin (BSA) (Perkin-Elmer, Wellesley, MA).
?5 Subunit Knockout Mice (?5?/?)
129/svJae background ?5 subunit knockout mice were generated and
maintained in our laboratory as previously described (18).
IgG2b isotype mouse monoclonal antibody against ?v?5 was raised
and characterized as described in Results (Figure 1).
IR Lung Injury Model
Sprague-Dawley rats (300–500 g) (Charles River Laboratories, Wilming-
anesthesia with a tidal volume of 6 ml/kg, positive end-expiratory pres-
sure (PEEP) of 10 cm H2O, and 100% oxygen (Model 683; Harvard
Apparatus Co., Holliston, MA). A median sternotomy was performed,
heparin (200 IU) was injected into the right ventricle, and cannulas
were placed in the pulmonary artery and left ventricle. Unilateral lung
IR was induced by right pulmonary artery ligation for 30 min followed
by release and reperfusion for 3 h. Rats were treated with antibodies
intravenously immediately before the experiment (4 mg/kg) or with
adenovirus intramuscularly 7 d before the experiment (1010pfu/kg).
Previous studies have determined this to be the peak time for soluble
VEGFRII secretion from the liver (19). Contralateral lungs served as
Mice were transtracheally intubated and ventilated with a high tidal
volume of 20 ml/kg at a rate of 48 breaths/min (without PEEP) for
4 h using a mouse ventilator (Model 683; Harvard Apparatus Co).
(250 mg/ml) intraperitoneal injections (100 ?l/20 g) with equal volume
injections of normal saline in matched animals. Matched nonventilated
mice were administered equal volumes of anesthesia and saline to serve
as baseline controls. Mice were administered antibodies by intraperito-
neal injection 24 h before the experiment (4 mg/kg) or adenovirus
intramuscularly 7 d before the experiment (1010pfu/kg) (19). TGF-?-
RII-Ig (25 ?g in 100 ?l sterile saline) was administered intravenously
immediately before initiation of ventilation. Lungs were harvested
immediately after 4 h of ventilation for lung vascular leak assay
Quantification of VEGF-Induced Vascular Leak
Vascular leak was studied 7 d after intramuscular administration of 1010
pfu/kg AdVEGFRII-Ig adenovirus (19) by measuring the extravasation
of Evan’s blue dye (30 mg/kg in 50 ?l per mouse). After 5 min, vascular
leak was induced by dermal injection of VEGF (100 ng in 10 ?l normal
saline) into mouse ears. After 1 h, 4 mm punch sections around the
VEGF injection site were harvested and formamide-extracted dye was
quantified as absorbance at 610 nm with a Spectra Max 190 Spectro-
photometer (Molecular Devices, Sunnyvale, CA).
Lung Vascular Leak and Protein Permeability
0.5 ?Ci of
administered intraperitoneally 4 h before lung harvest to ensure ade-
quate distribution. After each experiment, ablood sample was obtained
to measure the hemoglobin concentration and the water-to-dry weight
ratio of blood for the extravascularplasma equivalents (EVPE) calcula-
tion. Lungs (left for IR, and bilateral for VILI) were homogenized and
the extravascular lung water determined by calculating the water-to-
dry weight ratio using the following equation: W/D ? Qwet/Qdry, in which
Qwetisthe difference between the watercontent ofthe lung homogenate
and the water content of the blood in the lung, and Qdryis the dry lung
weight calculated as the weight of the lungs minus the blood and water
volumes in the lung. Lung endothelial permeability to albumin, ex-
pressed as EVPE in ml, calculated using the following equation: EVPE
? (CH– (CPend? QB))/CPave.CHrepresents the
homogenized lung, CPendrepresents the counts/min/g in plasma at the
end of the experiment, and CPaverepresents the average counts/min/g
in the plasma samples at the end of the experiment. QBis the blood
from wet and dried lung homogenates (19, 20). Counts were measured
on a Wizard ? counter (Perkin-Elmer). Control lungs included the
contralateral nonischemic, nonreperfused right lung for IR, and lungs
from nonventilatedmice forVILI. Baseline lungs forIR wereharvested
from animals not subject to pulmonary artery ligation.
125I-labeled albumin in 300 ?l sterile normal saline was
125I counts/min/g in the
Human pulmonary artery endothelial cells (HPAECs) (passages 3–9)
(Clonetics, Walkersville, MD) were maintained in EBM-2 basal endo-
thelial media supplemented with EGM-2 supplemental aliquots
(Clonetics). Bovine pulmonary arteryendothelial cells (BPAECs) (pas-
sages all ? 10) (CCL-209, ATCC) and mouse pulmonary endothelial
cells (see Isolation of Primary Mouse Endothelial Cells from ?5
Subunit Knockout Mice below) were cultured in Dulbecco’s minimal
essential (DME)/F-12 medium supplemented with 20% fetal bovine
serum (FBS),50 mg/liter ofendothelial mitogen (Biomedical Technolo-
gies, Stoughton, MA), and 10,000 U/liter of heparin. Cells were main-
tained on Corning polystyrene culture dishes (Fisher Scientific, Pitts-
burgh, PA) coated with type VI collagen (Sigma) and seeded onto
surfaces pre-coated with vitronectin (Upstate Biotechnology, Char-
lottesville, VA), fibrinogen (Calbiochem), or recombinant TGF-?1
latency-associated peptide (LAP) (21) or onto collagen-coated transwells
(Corning, Corning,NY) as required for individual experiments. Human
SW480 cells (CCL-228, ATCC) were infected with a retrovirus to ex-
press full-length integrin ?3 (pBABE-puro-?3) or transfected with the
plasmid vector pcDNA1-neo-?6 to express full-length ?6 (SW480-?3
and SW480-?6 cells). SW480-?8 cells were a generous gift from Steve
Nishimura, University of California, San Francisco. SW480 cells were
maintained in DMEM supplemented with 10% FBS and an appropriate
selection marker (Geneticin [G418, Life Technologies, Inc., Carlsbad,
CA] or puromycin [Calbiochem]).
Cell Adhesion Assay
Cells were allowed to adhere for 1 h to wells coated with a range of
saline, or the tested blocking antibody. Bovine serum albumin (BSA)-
coated wells served as nonadhesion controls. Plates were then spun
topside down at 40 ? g to remove nonadherent cells, and the remaining
cells were fixed with formalin, stained with crystal violet, and quantified
by absorbance (595 nm).
Isolation of Primary Mouse Endothelial Cells from ?5 Subunit
Lung tissue was collected from ?5 subunit knockout mice, pureed,
digested with 0.1% collagenase A, filtered through 10-?m nylon mesh,
centrifuged, and plated. At 16 h, negative selection was performed
with M-450 Dynabeads pre-conjugated with anti-Fc? receptor II/III
antibody. Positive selection with Dynabeads pre-conjugated with anti-
ICAM-2 antibody was performed on Days 3 and 7. To assess purity,
Su, Hodnett, Wu, et al.: Integrin ?v?5 and Lung Vascular Permeability 379
Figure 1. Characterization of anti-?v?5
antibody (ALULA). (A) Mouse L fibroblast
cell adhesiontoa rangeof concentrations
of vitronectinwasdeterminedin thepres-
ence of a range of concentrations of
ALULA. Data shown are the means ? SE,
n? 3.(B) SW480cellswere metabolically
labeled with35S-methionine, lysed in 1%
Triton, and immunoprecipitated either
without antibody, with ALULA, or with
the commercially available mouse anti-
human ?v?5 antibody, PIF6 (84). Precipi-
tated proteins were separated by nonre-
ducing SDS-PAGE and developed by
autoradiography. (C) SW480 cell adhe-
sion to a range of concentrations of
vitronectin was determined in the pres-
ence of a range of concentrations of
ALULA. Data shown are the means ?
SE, n ? 3. (D) Excess P1F6 completely
inhibited binding of ALULA to mock-
transfected SW480 cells (Mock), which
express ?v?5 as their only ?v-containing
integrin. P1F6 competitively excludes
ALULA from recognition binding sites in
SW480 cells transfected with ?3, ?6, and
?8 (SW480-?3, SW480-?6, SW480-?8).
Cells were assessed by flow cytometry
with antibodies specific for ?3 (AP-3), ?6
(3G9), or ?8 (37E.1) (solid histograms).
Biotinylated ALULA (Biotin-ALULA) anti-
body binding was assessed (shaded histo-
itive binding (100 ?g/ml). Controls (no
primary antibody) are shown as open his-
tograms. (E) SW480-?3 cell adhesion to
a range of concentrations of fibrinogen
was determined in the presence of ALULA
(10 ?g/ml). ?v?3-specific blockade of
blocking antibody (LM609). Data shown
are the means ? SE, n ? 3. (F) ?5 knock-
out mouse pulmonary endothelial cells
nary endothelial cells expressing full-
length human ?3 (?5?/?[?3]) were as-
sessed with flow cytometry using ALULA
and anti-?v?3 antibody (AP-3). (G)
SW480-?6 cell adhesion to a range of
concentrations of TGF-?1-latency-associated peptide (LAP)was determined in the presenceof ALULA (10 ?g/ml).?v?6-specific blockadeof adhesion
was achieved using ?6-blocking antibody (3G9). Data shown are the means ? SE, n ? 3. (H) SW480-?6 cells were assessed with flow cytometry
with biotinylatedanti-?v?6 antibody(Biotin-3G9) at different concentrations (2 ?g/ml, 0.2 ?g/ml, and 0.02 ?g/ml)(solid histograms)and reassessed
with P1F6 competitive binding (100 ?g/ml). Controls (no primary antibody) are shown as open histograms.
cells were analyzed for expression of ICAM-2 and PECAM by flow
cytometry (FACSort; Becton Dickinson, Franklin Lakes, NJ) and
CD34 and VE-cadherin by immunocytochemistry.
Conditional Immortalization of ?5 Subunit Knockout and
Wild-Type Mouse Pulmonary Endothelial Cells and ?5
Primary endothelial cells were transfected with the tsA58 SV40 large
and small T antigen genome (pUC18-tsA58 SV40) (generous gift from
Jiyue Zhu, Penn State College of Medicine, Hershey, PA) using Lipo-
fectamine 2000 (Invitrogen, Carlsbad, CA) and repeatedly passaged
for over 1 mo at 33?C to select for immortalized cells. Cells were
incubated at 39?C for 24 h before all described experiments and before
assessment for purity with endothelial cell markers (ICAM-2, VE-
cadherin, PECAM, and CD-34). Immortalized cells were infected with
retrovirus expressing full-length ?5 (pWZL-?5) generated by sub-
cloning ?5 cDNA (Clone 5.1; ATCC) (22) into a pWZL-blast2 vector
containing a blasticidin resistance gene.
Assay of Transendothelial Albumin Flux
Cells were seeded onto 6.5-mm collagen-coated PFTE membrane
to confluence. Cells were incubated with antibodies (10 ?g/ml) for 1 h
and then stimulated with VEGF (30 ng/ml), TGF-? (10 ng/ml), or
thrombin (10 U/ml) for 1 h.14C-BSA (0.005 ?Ci) (Perkin-Elmer) was
applied toeach uppercompartmentfor 1 h at 37?C, afterwhich contents
380 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 362007
from the lower compartment were collected and counted with an LS
6500 Multi-Purpose Scintillation Counter (Beckman, Fullerton, CA).
Only monolayers retaining ? 97% of tracer at baseline were studied.
Stress Fiber Visualization
Cells were grown on collagen-coated glass coverslips to confluence over
4 d. Serum-starved cells (12 h) were pre-treated with either control
antibody or ALULA for 1 h, then stimulated with respective agonists
(VEGF [30 ng/ml], TGF-? [10 ng/ml], or thrombin [10 U/ml]) for
10 min. Cells were then fixed with 3.7% paraformaldehyde for 10 min,
permeabilized with 0.5% triton X-100, then stained with rhodamine
phalloidin (Molecular Probes, Carlsbad, CA), mounted, and imaged
using a Leica DM5000B microscope equipped for epifluorescence.
RhoA Activation Assay
Cells were grown on collagen-coated 100-mm culture dishes, serum-
col (Upstate Biotechnology). After centrifugation, lysate supernatants
were incubated with agarose beads conjugated with rhotekin Rho-
binding domains (RBD) that recognize only GTP-bound active RhoA.
RhoA was detected from pulled-down product by Western analysis
using anti-RhoA antibodies (Upstate Biotechnology). GTP?S and
GDP-spiked lysates served as positive and negative controls.
?v?5 Antibody Characterization
Therefore,we raised amonoclonal antibodyagainst mouse ?v?5
(ALULA) by immunizing?5 subunitknockout micewith mouse
L fibroblasts expressing ?v?5. We confirmed that ALULA func-
tionally blocked mouse L cell adhesion to the ?v?5 ligand
vitronectin (Figure 1A). ALULA has an identical immunopre-
cipitation profile to the previously validated anti-?v?5 antibody
P1F6 (23) (Figure 1B), and inhibits adhesion of human SW480
cells to vitronectin (Figure 1C). To exclude ALULA recognition
of other?v-containing integrins, we performedbinding site com-
petition assays using the anti-?v?5 antibody, P1F6. Excess P1F6
completely inhibited binding of ALULA to mock-transfected
SW480cells,which express?v?5astheironly ?vintegrin(Figure
1D). ALULA binding to SW480 cells transfected with human
?3, ?6, or ?8 (SW480-?3, -?6, and -?8) was similarly inhibited
by P1F6 (Figure 1D). Furthermore, ALULA did not inhibit
adhesion of SW480-?3 to the ?v?3 ligand fibrinogen (Figure
1E) and did not recognize ?v?3 in ?5 knockout pulmonary
endothelial cells transfected with full-length human ?3 (Figure
1F). ALULA was also incapable of inhibiting adhesion of
SW480-?6 cells to the ?6 ligand TGF-?1 LAP (Figure 1G).
Anti-?v?6 antibody (3G9) binding to SW480-?6 cells was not
affected by excess P1F6 at any dilution of 3G9 tested (Figure
1H). ALULA also did not affect SW480-?8 cell adhesion to
TGF-?1-LAP, but this finding is not straightforward to interpret
because of the lack of available ?v?8 antibodies that inhibit
cell adhesion for use as a positive control (data not shown). In
addition to mouse and human ?v?5, ALULA was shown to
recognize bovine ?v?5 by flow cytometry using bovine pulmo-
nary artery endothelial cells (BPAECs) (data not shown).
?v?5 Regulates Lung Vascular Permeability in a Model of
Lung IR-Induced ALI
We initially chose to study IR-induced lung injury in rats (24)
since VEGF had been implicated as a possible mediator of in-
creased lung vascular permeability after IR (25, 26), and since
Figure 2. ?v?5 regulates lung vascular permeability in a rat model of
IR-induced ALI. Rat lungs were subjected to unilateral left lung ischemia
(30 min) followed by reperfusion (3 h) (open bars). Rats were treated
with ALULA or an IgG2b isotype control antibody (Control Ab), VEGF
receptor II-Ig chimera adenovirus (AdVEGFRII-Ig), or green fluorescent
protein (GFP) adenovirus control (AdGFP). Lung vascular permeability is
reported as extravascular I125-albumin leak normalized to leak measured
from lungs harvested from animals not subject to pulmonary artery
nonischemic, nonreperfused lung in each individual animal served as
its control lung (Control; solid bars). Data shown are the means ? SE,
n ? 5. *P ? 0.049 for rats treated with ALULA compared with those
treated with Control Ab; **P ? 0.002 for rats treated with AdVEGFRII-
Ig compared with those treated with control AdGFP.
permeability, as measured by parenchymal extravasation of an
I125-albumin intravascular tracer, which was completely blocked
by systemic administration of ALULA (Figure 2). To determine
whether IR-induced lung vascular permeability was indeed de-
pendent on VEGF, we used an adenovirus expressing a VEGF
receptor II-IgG chimera (AdVEGFRII-Ig) that had previously
(27). Administration of AdVEGFRII-Ig blocked IR-induced
lung vascular permeability, whereas a control adenovirus ex-
pressing green fluorescent protein (AdGFP) did not (Figure 2).
?v?5 Regulates Lung Vascular Permeability in VILI
To determine whether the protective effect of blocking ?v?5
with ALULA could be generalized to other models of increased
lung vascular permeability, and to take advantage of the avail-
ability of ?5 knockout mice (18), we used a mouse model of
VILI in which lung vascular permeability was induced by four
hours of mechanical ventilation at a high tidal volume (20 ml/kg)
(28). This model produced a robust increase in lung vascular
permeability,whichwas completelyblockedby ALULA(Figure
3A). ?5 subunit knockout mice were also completely protected
from increased lung vascular permeability (Figure 3A). How-
ever, in contrast to our results from the IRmodel, AdVEGFRII-
Ig did not block lung vascular permeability induced by VILI.
Adequate blockade of VEGF with AdVEGFRII-Ig was con-
firmed by the absence of dermal vascular leak after intradermal
VEGF injection (Figure 3B). Since previous reports have associ-
ated the multifunctional cytokine TGF-? with VILI (29, 30),
andourown previous workhasshown TGF-?to beanimportant
mediator of lung vascular permeability induced by other stimuli
saccharide ), we examined the role of TGF-? in our VILI
model. Administration of a recombinant TGF-? receptor II IgG
chimera (TGF-?RII-Ig) significantly inhibited lung vascular per-
meability in VILI (Figure 3A).
?v?5 Regulates Pulmonary Artery Endothelial Permeability
Induced by Diverse Mediators of ALI
Our in vivo data suggested that the protective effects of blocking
?v?5 were not restricted to effects on VEGF-induced increases
in vascular permeability. Since VEGF and TGF-? activate dis-
tinct families of receptors and trigger different initial signaling
Su, Hodnett, Wu, et al.: Integrin ?v?5 and Lung Vascular Permeability381
Figure 3. ?v?5 regulates lung vascular permeability in VILI. (A) Mice
were treated with ALULA orisotype controlantibody (ControlAb), VEGF
receptor II-Ig chimera adenovirus (AdVEGFRII-Ig) or green fluorescent
protein (GFP) adenovirus control (AdGFP), or TGF-? receptor II IgG
chimera (TGF-?RII-Ig) or its vehicle control before ventilation at high
tidal volume (20 ml/kg) for 4 h. Lung vascular permeability is reported
as extravascular I125-labeled albumin leak normalized to baseline. Data
shown are the means ? SE, n ? 6. *P ? 0.026 for mice treated with
ALULA compared with those treated with Control Ab; **P ? 0.021 for
?5 knockout compared with wild-type (WT) control mice; ***P ? 0.027
for mice treated with TGF-?RII-Ig compared with those treated with
vehicle control. Baseline lung vascular permeability was measured from
permeability. Blockade of VEGF-induced vascular permeability by
AdVEGFRII-Ig was determined by measurement of extravasation of
Evan’s blue dye. ?5 knockout or WT mice that had been infected with
either AdVEGFRII-Ig or AdGFP were injected intravenously with Evan’s
blue dye. Vascular leak was induced by dermal injection of VEGF (solid
bars). Open bars, saline. Total Evan’s blue dye content in dermal biopsy
sections was determined by formamide extraction and measurement
of optical density (O.D.) absorbance at 610 nm. Data shown are the
means ? SE, n ? 6. *P ? 0.001 for mice treated with AdVEGFRII-Ig
compared with those treated with control AdGFP; **P ? 0.037 for ?5
knockout compared with WT control mice.
tory role downstream of multiple agonist pathways. To test this
hypothesis, we studied the effects of VEGF, TGF-?, and also
the serine protease thrombin, on pulmonary endothelial cell
barrier permeability using a C14-albumin flux assay. Thrombin
has been extensively studied for its potent effects on increasing
pulmonary endothelial permeability (13, 32–36). We stimulated
both bovinepulmonary artery endothelialcell(BPAEC) (Figure
4A) and human pulmonary artery endothelial cell (HPAEC)
(Figure 4B) monolayers with VEGF, TGF-?, and thrombin and
found that dose-dependent increases in transendothelial C14-
albumin flux in response to each agonist were blocked by
Genetic Absence of ?v?5 in Pulmonary Endothelial Cells
Protects against Permeability Induced by Diverse Mediators
To confirm specificity of this effect to ?v?5, we used ?5 knockout
pulmonary endothelial cells isolated from ?5 knockout mice. Pri-
mary mouse ?5 knockout cells were conditionally immortalized
by retroviral transfer of a temperature-sensitive SV40 large and
small T antigen transgene. ?5 reconstitution was performed by
retroviral transfer of full-length human ?5 into the immortalized
(Figures 4C–4D). ?5 knockout cells were completely resistant
to increases in endothelial permeability induced by all three
agonists (Figure 4E). As seen in both BPAECs and HPAECs,
VEGF, TGF-?, and thrombin each induced dose-dependent in-
creases in monolayer permeability in ?5 reconstituted mouse
pulmonary endothelial cells (Figure 4E). ?5 reconstituted cells
incubated with ALULA were resistant to agonist-induced per-
meability changes (Figure 4E).
?v?5 Regulates Agonist-Induced Stress Fiber Formation
Filamentous (F)-actin can be induced to polymerize to form
transcytoplasmic cables that generate tension between cell–cell
junctions and focal adhesions on the extracellular matrix. Sub-
sequent disruption of cell–cell junctions can lead to increased
layers of BPAECs, treated with ALULA or control antibody,
with the agonists VEGF, TGF-?, and thrombin. As has pre-
viously been reported (10–16), each of these agonists caused
dramatic increases in actin stress fiber formation. Further-
more, in each case, this effect was markedly attenuated by ?v?5
blockade with ALULA compared with treatment with control
antibody (Figure 5A).
Agonist-Induced Stress Fiber Formation and Permeability in
BPAECs Are Blocked by RhoA Kinase Inhibition
The RhoA family of GTPases has been shown to be a critical
regulator of actin stress fiber formation (37–39) as well as an
important regulator of increased endothelial permeability (32,
33, 40–48). Each of the three agonists we used for these studies
has previously been shown to activate RhoA (45, 49–59). We
confirmed these findings by showing that inhibition of RhoA-
activated kinase (ROCK) (immediate downstream effector of
RhoA) prevents VEGF-, TGF-?-, and thrombin-induced stress
fiber formation (Figure 5A). Furthermore, we confirmed that
agonist-induced changes in permeability were completely blocked
by ROCK inhibition (Figure 5B).
Agonist-Induced RhoA Activation in BPAECs Is Unaffected by
To determine whether ?v?5 might regulate agonist-induced
tion, we used a rhotekin RhoA-binding domain (RBD)-based
active RhoA assay, to assess global agonist-induced increases in
cellular RhoA activity. Previous studies have shown that RhoA
is activated by thrombin, VEGF, and TGF-? (45, 49–59). We
found robust RhoA activation by thrombin in BPAECs that
was completely unaffected by ?v?5 blockade (Figure 5C). This
finding suggests that ?v?5 contributes to RhoA and ROCK-
mediated induction of stress fiberformation and increased endo-
thelial permeability by acting downstream of RhoA.
Despite numerous advances in our understanding of the patho-
physiology of ALI, specific molecular mechanisms responsible
for this clinical syndrome remain poorly understood. In this
for integrin ?v?5 in the development of an important pathologic
382 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 362007
Figure 4. ?v?5 regulates permeability of pulmonary endothe-
starved confluent BPAEC monolayers on Transwells were
incubated with ALULA or isotype control antibody (Control
Ab) 1 h before stimulation with VEGF, TGF-?, or thrombin at
the doses shown. Transendothelial leak was determined by
quent collection and scintillation counting of basolateral well
contents after 1 h. Data shown are the means ? SE, n ? 3.
*P ? 0.013 for VEGF (30 ng/ml); **P ? 0.018 for TGF-?
(10 ng/ml); ***P ? 0.022 for thrombin (10 U/ml) for cells
treated with ALULA compared with those treated with Control
Ab. (B) Serum-starved confluent HPAEC monolayers on
Transwells were incubated with ALULA or Control Ab 1 h
before stimulation with VEGF, TGF-?, orthrombin at the doses
shown. Data shown are the means ? SE, n ? 3. *P ? 0.038
for VEGF (30 ng/ml); **P ? 0.041 for TGF-? (10 ng/ml);
***P ? 0.050 for thrombin (10 U/ml) for cells treated with
ALULA compared with those treated with Control Ab. (C) ?5
subunit knockout pulmonary endothelial cells (?5?/?) were
assessed for endothelial markers by immunocytochemistry
try (platelet endothelial cell adhesion molecule [PECAM] and
intracellular adhesion molecule [ICAM]-2). Positive immuno-
cytochemistry staining is shown in white. (D) ?5 subunit
knockout (?5?/?) and ?5 reconstituted (?5?/?[?5 Recon])
mouse pulmonary endothelial cells were assessed for expres-
sion of ?5 by flow cytometry with ALULA. Flow cytometry
diagrams represent cells incubated with PBS and secondary
antibody alone(solidhistograms) orwith selectedprimaryanti-
bodies (open histograms). (E) Serum-starved ?5 knockout
endothelial cell monolayers seeded onto Transwells were as-
sessed for monolayer permeability. ?5 reconstituted cells were
incubated with ALULA or a Control Ab (10 ?g/ml) 1 h before
stimulation of both cell types with VEGF, TGF-?, or thrombin
at the doses shown. Data shown are the means ? SE, n ? 3.
*P ? 0.006 for VEGF (30 ng/ml); **P ? 0.004 for TGF-?
(10 ng/ml); ***P ? 0.014 for thrombin 10 U/ml for ?5?/?(?5
Recon) cells treated with Control Ab compared with ?5?/?
cells.†P ? 0.009 for VEGF (30 ng/ml);††P ? 0.004 for TGF-?
(10 ng/ml);†††P?0.011 forthrombin(10U/ml)for?5?/?(?5
Recon) treated with ALULA compared with those treated with
hallmark of ALI–increased lung vascular permeability. Further-
more, ?v?5 regulates increased permeability in human, bovine,
and murine pulmonary endothelial cells induced by a range
of different edemagenic agonists including VEGF, TGF-?, and
network of proinflammatory mediators, produced locally in the
lung (by fibroblasts, inflammatory, epithelial, and endothelial
cells), or derived from extrapulmonary sources, are thought to
initiate and amplify the inflammatory response in ALI (31, 60, 61).
Our studies have also identified ?v?5 as a specific regulator
of induction of actin stress fibers, a well-described contributor
finding suggests that ?v?5 might regulate changes in vascular
eton. Integrins are known to be principal components of focal
adhesions—multimolecular structures that link the extracellular
matrix (ECM) to the intracellular cytoskeleton. Several reports
to endothelial barrier function, but the precise molecular basis
for this regulation remains unclear (62–65). Butler and coworkers
recently reported that isolated focal adhesion complexes can
initiate actin polymerization of actin monomers de novo (66).
Moreover, these investigators showed that actin polymerization
is dependent on physical clustering of integrins to focal adhesion
structures (66). Additional studies are required to identify the
molecular mechanisms by which ?v?5 regulates stress fiber
Physical passage of solutes through the endothelial barrier is
thought to occur via paracellular pathways or through receptor-
activated transcytosis (67, 68). The functional relevance, relative
contribution,andmoleculardeterminants ofthese distinctmech-
anisms remain incompletely understood, but it has been sug-
gested that direct modification of the actin cytoskeleton in endo-
thelial cells is important for increasing paracellular permeability.
One frequentlycited modeldescribes paracellulargapformation
as a consequence of imbalanced competition between cytoskele-
tal, adhesive cell–cell and cell–matrix forces (10, 16, 47, 69). In
this model, F-actin polymerizes and bundles into morphologi-
cally distinct “stress fibers”. Actomyosin-mediated generation
of tension leads to alteration of cell shape and formation of
paracellular gaps. Stress fibers have been shown to form in endo-
thelial cells stimulated by several vasoactive mediators (11–15,
Su, Hodnett, Wu, et al.: Integrin ?v?5 and Lung Vascular Permeability 383
Figure 5. Agonist-induced
formation in bovinepulmonary artery en-
dothelial cells is attenuated by ?v?5
blockade and ROCK inhibition, but ?v?5
blockade has no effect on RhoA activity.
pretreated with either isotype control an-
tibody (Control Ab) (10 ?g/ml), ALULA
(10 ?g/ml), or ROCK inhibitor (Y-27632)
(10 ?M) for 1 h, then stimulated with ago-
nists VEGF (30 ng/ml), TGF-? (10 ng/ml),
and thrombin (10 U/ml) for 10 min. Cells
were fixed, permeabilized, and stained
with rhodamin-phalloidin. (B) Serum-
starved confluent BPAEC monolayers on
Transwells were incubated with ROCK in-
hibitor (Y-27632)(10 ?M) orvehicle con-
trol for 1 h before stimulation with VEGF,
TGF-?, or thrombin at the doses shown.
Transendothelial leak was determined
by application of a C14-albumin tracer to
the apical well and subsequent collection
and scintillation counting of basolateral
well contents after 1 h. Data shown are
the means ? SE, n ? 3. *P ? 0.001 for
VEGF (30 ng/ml); **P ? 0.005 for TGF-?
(10 ng/ml); ***P ? 0.003, for thrombin
(10 U/ml) for cells treated with Y-27632
compared with those treated with vehi-
cle. (C) Total cellular RhoA activation in
was assessed using BPAECs pre-treated
with either Control Ab (10 ?g/ml) or
ALULA (10 ?g/ml), that were then stimu-
lated for 2 min with thrombin (10 U/ml).
rosebeads conjugatedwith rhotekinRho-
binding domains (RBD) that recognize
only GTP-bound active RhoA (immuno-
tected from pulled-down product by im-
munoblot (IB) using anti-RhoA antibody
(RhoA). Total lysate samples not incu-
bated with rhotekin beads were immu-
noblotted with anti-RhoA antibody and
70, 71), including VEGF, TGF-?, and thrombin (45, 49–59).
Although our studies did not directly distinguish between para-
cellular and transcellular pathways, the parallel ability of ?v?5
to regulate both stress fiber formation and transendothelial flux
suggests that in our system the paracellular pathway may be the
Use of cells derived from proximal pulmonary macrovascular
endothelium is a limitation to our studies. Microvascular endo-
ically relevant model of pulmonary capillary leak and many
studies have detailed significant physiologic differences between
lung cells from microvascular and macrovascular bed origins
(72–78). Previously, the ?v?5-specific antibody P1F6 was shown
to have no effect on ligand-induced increases in lung capillary
hydraulic conductivity (79). Our studies are different because
we have focused on agonist-induced permeability events, rather
than on effects of integrin ligand binding alone. In fact, we found
no effect of ?v?5 blockade on baseline permeability or on lung
cular endothelial and alveolar epithelial cell co-culture systems
(80), or perhaps capillary split-drop techniques (79, 81) would
be necessary to address the important issue of what role ?v?5
might play in regulating capillary permeability.
The model of VILI we used uses relative tidal volumes sub-
Therefore, results of the current study cannot be directly extrap-
olated to suggest that ?v?5 blockade would diminish increased
permeability induced by volutrauma in mechanically ventilated
patients. Nonetheless, this model is widely used and likely does
reflect the effects of excess stretch on alveolar units. Determina-
tion of the direct relevance of our findings to patients with VILI
will need to await clinical studies with drugs designed to target
Several important unanswered questions remain, including
how actin stress fiber formation is regulated by ?v?5 ligation.
Our observations that ?v?5 antibody produced identical results
to both ?5 knockout miceand ?5 knockout cells strongly suggest
384 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 362007
that theantibody exerted itseffectby specificallyinhibiting ?v?5
function, rather than as a result of other antibody–integrin inter-
actions. VEGF, TGF-?, and thrombin activate different families
of receptors (tyrosine kinase, serine-threonine kinase, and G
protein coupled, respectively) that initiate distinct proximal sig-
naling pathways. It will be important to determine how these
diverse pathways converge on ?v?5, and to identify common
signaling intermediates. An exampleof sucha signaling interme-
diate might be the RhoA small GTPase, which has been shown
to be activated downstream to a variety of different agonist
pathways (45, 49–59), and to be both a critical regulator of
actin stress fiber formation (37–39) and increased endothelial
permeability (32, 33, 40–48). Our findings in this report suggest
that total cellular RhoA activation is not directly affected by
?v?5 blockade, implying that RhoA activation occurs upstream
While we have focused on disruption of the pulmonary endo-
thelial cell barrier as the main target of ?v?5 effects, there are
alternative targets to consider. In vivo increases in lung vascular
ple cell types, including leukocytes and epithelial cells, as well
as endothelial cells (82). Since ?v?5 is widely expressed, it is
possible that ?v?5-mediated effects on other cell types could
as models of increased lung vascular permeability. Relevance
to ALI, and even to their specific clinical correlates may be
questioned, for example, with experimental ischemia and reper-
fusion times (for IR) and brief relative ventilation periods and
extreme tidal volume settings (for VILI). The complexities of
ALI mandate that other models be tested. Ultimate proof of
relevance will only come from clinical studies in patients at risk
for or affected by ALI.
Finally, although our results demonstrate an important role
ity, they do not identify the relevant in vivo ligand. Our cell
culture studies were performed by seeding cells onto nonspecific
collagen substrates in the presence of fetal calf serum, a rich
overseveral days.This protocolallowed ampletimeforvitronec-
tin to bind to the cells and substrate and for the cells to secrete
specifically recognizes ?v?5 and blocks adhesion to vitronectin
in vitro. However, it is certainly plausible that, in vivo, other
ligands are critical for the functions we have described. While
vitronectin knockout mice have been observed to be viable and
healthy (83), and therefore, would be a good model system to
determine relevance of vitronectin, these studies might poten-
tially be confounded by effects exerted by the integrin ?v?3,
which shares vitronectin as a common ECM protein ligand.
Despite these gaps in our current understanding, the findings
reported here have potential clinical relevance. Given the robust
regulatory effects of blocking ?v?5 in two quite different in vivo
nary endothelial permeability response to multiple biologically
relevant agonists, ?v?5 appears to be an attractive therapeutic
target for ALI, a substantial cause of morbidity and mortality
that is currently largely untreatable.
Conflict of Interest Statement: G.S. does not have a financial relationship with a
commercial entity that has an interest in the subject of this manuscript. M.H.
does not have a financial relationship with a commercial entity that has an interest
in the subject of this manuscript. N.W. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. A.A.
does not have a financial relationship with a commercial entity that has an interest
in the subject of this manuscript. C.K. does not have a financial relationship with
a commercial entity that has an interest in the subject of this manuscript. M.G.
does not have a financial relationship with a commercial entity that has an interest
in the subject of this manuscript. X.Z.H. does not have a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
J.K.K. does not have a financial relationship with a commercial entity that has an
interest in the subject of this manuscript. J.A.F. does not have a financial relation-
ship with a commercial entity that has an interest in the subjectof this manuscript.
M.A.M does not have a financial relationship with a commercial entity that has
an interest in the subject of this manuscript. D.S. is co-owner of a filed patent
(pending) covering blockade of integrin ?v?5 for the treatment of acute lung
injury. He also has had a sponsored research agreement with BiogenIdec to cover
work on anti-integrin antibodies and acute lung injury for $150,000/year (total
costs) since January 2002. J.-F.P. does not have a financial relationship with a
commercial entity that has an interest in the subject of this manuscript.
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