Involvement of c-Src in diperoxovanadate-induced endothelial
cell barrier dysfunction
Shu
Shi,
Joe G. N.
Garcia,
Shukla
Roy,
Narasimham L.
Parinandi, and
Viswanathan
Natarajan
Division of Pulmonary and Critical Care Medicine, Johns Hopkins
University School of Medicine, Baltimore, Maryland 21224
 |
ABSTRACT |
Reactive oxygen species (ROS)
generated by activated leukocytes play an important role in the
disruption of endothelial cell (EC) integrity, leading to barrier
dysfunction and pulmonary edema. Although ROS modulate cell signaling,
information remains limited regarding the mechanism(s) of ROS-induced
EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model
agent to explore the role of tyrosine phosphorylation in the
regulation of EC barrier function. DPV disrupted EC barrier function in
a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and
PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier
dysfunction. Consistent with these results, DPV-induced Src activation
was attenuated by PP-2. Furthermore, DPV increased the association of
Src with cortactin and myosin light chain kinase, indicating their
potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src
mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These
studies provide the first direct evidence for Src involvement in
DPV-induced EC barrier dysfunction.
non-receptor tyrosine kinases; tyrosine phosphorylation; vascular
permeability
 |
INTRODUCTION |
THE ENDOTHELIUM
FUNCTIONS as a semiselective barrier between the plasma and the
interstitium to circulating bioactive agents, inflammatory cells, and
macromolecules (6, 8, 9, 38). Maintenance of this vascular
barrier represents a critical physiological process for vessel
homeostasis and organ function. During acute lung injury, however,
impaired barrier function leads to the exudation of fluids and proteins
into the interstitium (29), alveolar flooding, and
subsequent derangements in lung compliance and gas exchange, a
characteristic feature of acute respiratory distress syndrome
(2). Development of acute lung injury in many settings has
been tightly coupled to the activation of polymorphonuclear leukocytes
in the lung microvasculature, with the subsequent release of proteases,
inflammatory mediators, and reactive oxygen and nitrogen intermediates
(14, 15, 54). As a result of its extensive surface area,
the pulmonary endothelium is a prime target for the inflammatory
mediators and reactive oxygen species (ROS), resulting in cellular
damage and barrier dysfunction (3, 27).
ROS generated during ischemia-reperfusion lung injury or the
exogenous addition of either hydrogen peroxide
(H2O2) or xanthine/xanthine oxidase to
endothelial cell (EC) monolayers resulted in morphological, biochemical, and physiological perturbations such as barrier
dysfunction (24, 26, 41, 44). Although the
mechanism(s) of ROS-induced EC barrier dysfunction is not well
understood, earlier studies (5, 7, 27, 31, 49, 54)
suggested that in addition to potential ROS-induced cytotoxicity,
modulation of protein kinases or phosphatases and generation of
intracellular second messengers may be responsible for ROS-mediated
changes in vascular permeability. Exposure of bovine pulmonary artery
ECs (BPAECs) to ROS increased permeability to albumin (41)
that was dependent on protein kinase C (PKC) activation
(22) and increased Ca2+ availability
(39, 44). Similarly, inhibition of PKC with H-7 prevented
H2O2-induced pulmonary edema in isolated
perfused guinea pig lungs (20). In Madin-Darby canine
kidney cells, treatment with orthovanadate or pervanadate, potent
inhibitors of protein tyrosine phosphatases (PTPases), increased the
levels of phosphotyrosine proteins that colocalized with adherens
junction proteins, with disruption of cell junction-matrix contacts and
increased tight junction permeability (28, 46). A recent
study (42) in BPAECs suggested a role for protein tyrosine
phosphorylation in thrombin-induced EC contraction and permeability via
a non-receptor tyrosine kinase (TyK) that was sensitive to genistein.
Although these studies with vanadate, pervanadate, and thrombin
suggested a potential role for contractile and cytoskeletal proteins in
gap formation and paracellular transport, very little is known
regarding the mechanism(s) of ROS-induced endothelial barrier
dysfunction and signaling pathways that regulate the contractile and
tethering forces.
The aim of the present study was to determine the role of protein
tyrosine phosphorylation in endothelial barrier dysfunction and to
identify specific TyKs involved in the regulation of EC permeability.
We employed diperoxovanadate (DPV), a potent inhibitor of PTPases and
activator of TyKs (32, 37), as a model agent to
investigate the mechanism(s) of endothelial barrier dysfunction. Our
results in BPAECs demonstrate that DPV-mediated protein tyrosine phosphorylation is involved in permeability changes. Our data also show
for the first time that the activation and tyrosine phosphorylation of
the Src family of non-receptor TyKs regulate vascular permeability.
Furthermore, because DPV increased the association of Src with actin
binding protein, cortactin, and myosin light chain (MLC) kinase (MLCK),
these proteins may represent cytoskeletal targets involved in
DPV-mediated EC barrier dysfunction.
 |
METHODS |
Materials.
Minimum essential medium (MEM), H2O2,
sodium orthovanadate, nonessential amino acids, trypsin-EDTA,
penicillin-streptomycin, and fetal bovine serum were obtained from
Sigma (St. Louis, MO). BPAECs (passage 16)
were purchased from American Type Culture Collection (Manassas, VA).
Genistein, Brij 35 detergent (polyoxyethyleneglycol dodecyl
ether), and PP-2 were obtained from Calbiochem (San Diego, CA).
Endothelial cell growth supplement, affinity-purified monoclonal anti-phosphotyrosine antibody (4G10), Src cDNAs (pUSE src
wild type, pUSE src activated, pUSE src kinase
mutant, and empty vector), and monoclonal antibody to Src were obtained
from Upstate Biotechnology (Lake Placid, NY). An enhanced
chemiluminescence kit was from Amersham (Arlington Heights, IL).
Polyclonal antibody to Src and protein A/G plus agarose were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA).
[
-32P]ATP in 10 mM Tricine buffer (specific activity
6,000 Ci/mmol) was purchased from NEN (Boston, MA). Crystallized DPV
(potassium salt), prepared by mixing equimolar amounts of
H2O2 and sodium orthovanadate
(40), was kindly provided by Dr. T. Ramasarma (Indian
Institute of Science, Bangalore, India).
EC culture.
BPAECs cultured in MEM were maintained at 37°C in a humidified
atmosphere of 5% CO2-95% air (52) and grown
to contact-inhibited monolayers with a typical cobblestone morphology.
Cells from each primary flask were detached with 0.05% trypsin,
resuspended in fresh medium, and cultured on either polycarbonate
filters for permeability studies (36), 11-mm wells for
electrical resistance determinations (42), or 100-mm
dishes for Src immunoprecipitation experiments.
Measurement of EC permeability.
Macromolecule permeability of albumin across cultured EC monolayers was
performed as previously described (36). Briefly, the
system consisted of two compartments, upper (luminal) and lower
(abluminal), which were separated by a polycarbonate micropore membrane
filter (Nuclepore, Pleasanton, CA) on which the ECs were seeded to
confluence. For measurement of albumin flux, the lower compartment was
stirred continuously and kept at a constant temperature of 37°C by
use of a thermally regulated water bath. Medium 199 with 25 mM HEPES
was used in both compartments. Bovine serum albumin (4% final
concentration) complexed to Evans blue (EB) dye was added to the
luminal compartment, and samples were taken from the abluminal
compartment at 10-min intervals for the first 60 min to establish the
basal albumin clearance rate (baseline) and then for an additional 60- to 120-min period after each specific intervention. Transendothelial
cell albumin transport was determined by measuring the absorbance of EB
dye in abluminal chamber samples at 620 nm in a spectrophotometer (Vmax
Multiplate Reader, Molecular Devices, Menlo Park, CA). Albumin
clearance rates were calculated by linear regression analysis for
control and experimental groups.
Measurement of transendothelial cell electrical resistance.
Transendothelial cell electrical resistance was measured as described
earlier (42), with minor modifications. Briefly, in this
electrical cell-substrate impedance-sensing system (Applied Biophysics,
Troy, NY), ECs were cultured on a small gold electrode (10
4 cm2), and the culture medium was used as
the electrolyte. The total electrical resistance, measured dynamically
across the monolayer, was determined by the combined resistance between
the basal surface of the cell and the electrode, reflective of focal
adhesion, and the resistance between cells. Thus a change in electrical
resistance represents a change in cell-cell adhesion and/or cell-matrix
adhesion. A 1-V 4,000-Hz AC signal was supplied through a 1-M
resistor to approximate a constant-current source. Voltage and phase
data were stored and processed with a pentium 100-MHz computer that controlled the output of the amplifier and relay switches to different electrodes. Experiments were conducted only on wells that achieved >5,000
of steady-state resistance. Resistance is expressed as the
in-phase voltage (proportional to the resistance) that was normalized to the initial voltage and as a fraction of the
normalized resistance value, similar to that previously described
(42).
Preparation of cell lysates, immunoprecipitation, and immunoblot
analysis.
BPAECs grown in 100-mm culture dishes were stimulated with 5 µM DPV
for various times and rinsed with ice-cold PBS to stop stimulation. ECs
were lysed in modified radioimmunoprecipitation assay buffer (50 mM
Tris · HCl, pH 7.4, 150 mM NaCl, 1% Nonidet-40, 0.25% sodium
deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
Na3VO4, 1 mM NaF, 10 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 1 µg/ml of pepstatin), scraped off the
dishes, sonicated on ice with a probe sonicator (3 times for 15 s
each), and centrifuged at 14,000 rpm in a microfuge (4°C for 5 min), and protein concentrations of the supernatants were determined with a
Pierce protein assay kit. The supernatants, adjusted to 1 mg protein/ml
for immunoprecipitation, were precleared with protein A/G plus agarose
at 4°C for 60 min and incubated overnight with rabbit polyclonal
anti-Src antibody (Santa Cruz) at 4°C. Protein A/G plus agarose (20 µl) was then added, incubated for an additional 2 h at 4°C,
and centrifuged at 14,000 rpm in a microfuge for 5 min. The
precipitates were washed three times with ice-cold PBS and dissociated
by boiling in 1× SDS sample buffer for 5 min. The samples were then
analyzed on 10% SDS-PAGE gels (52), and densitometric
scanning of the blots was carried out with a Bio-Rad model GS-700
densitometer and quantified with Molecular Analyst software.
p60Src kinase activity.
To evaluate Src kinase activity, p60Src
immunoprecipitates were washed three times in ice-cold PBS as
described in Preparation of cell lysates, immunoprecipitation,
and immunoblot analysis and once in kinase assay buffer (50 mM HEPES, pH 7.5, containing 0.1 mM EDTA and 0.015% Brij 35 detergent). The kinase activity in the
p60Src-immunoprecipitated complex was determined
in a final reaction volume of 40 µl of 50 mM HEPES, pH 7.5, containing 0.1 mM EDTA, 0.015% Brij 35 detergent, 15 mM
MgCl2, 1 mM Na3VO4, 150 µM ATP, and 33 µCi of [
-32P]ATP with and without
raytide peptide, which acts as a substrate for Src tyrosine kinase
(42). The reaction mixture was incubated at 30°C for 30 min, and the reaction was terminated by the addition of 6× Laemmli
sample buffer or 10% phosphoric acid. The samples were boiled for 5 min and subjected to SDS-PAGE or spotted on P81 filter paper, washed
five times with 10% phosphoric acid, and counted in a scintillation counter.
EC transfection.
Src DNA plasmids (wild type, constitutively active, or dominant
negative) were transfected into ECs at 50-80% confluence with the
FuGENE 6 transfection reagent. The constitutively active Src cDNA
carries a Tyr-to-Ala substitution at amino acid 529; the dominant
negative cDNA has two point mutations, Lys-to-Arg substitution at
residue 296 and Tyr-to-Phe substitution at residue 528, whereas wild-type Src cDNA encodes the wild-type p60Src.
FuGENE 6 reagent (3-6 µl) was added directly into 100 µl of serum-free MEM and incubated for 5 min at room temperature, and the
diluted FuGENE 6 transfection reagent was added dropwise to a tube
containing Src cDNA (3 µg/ml). The contents of the tube were
incubated for 15 min at room temperature, transferred to 35-mm dishes
containing BPAECs (50-80% confluent), and incubated for 5 h.
At the end of the transfection, the Src cDNA-FuGENE 6 complex was
removed by aspiration, 2 ml of MEM containing 10% serum were added,
and the cells were incubated in 95% air-5% CO2 chamber
for 48 h. Protein expression was determined with Western blotting
48 h posttransfection.
Statistics.
Linear regression analysis was performed for determination of clearance
rates in individual wells with Epistat 2.0 public domain software, and
these slopes were then averaged from at least six determinations.
Paired t-test was used to compare pretreatment and
posttreatment slopes within the same control membrane or BPAEC chamber.
ANOVA with Student-Newman-Keuls test was used to compare means of
clearance rates of two or more different treatment groups. The level of
significance was taken to be P < 0.05 unless otherwise stated. Data are expressed as means ± SE.
 |
RESULTS |
DPV induced EC barrier dysfunction.
H2O2 increased albumin flux across EC
monolayers via a PKC-dependent pathway (45) and enhanced
protein tyrosine phosphorylation through modulation of TyKs and
PTPases (32). However, the role of protein
tyrosine phosphorylation in ROS-mediated EC barrier dysfunction is
poorly understood. To assess the potential involvement of protein
tyrosine phosphorylation in EC barrier regulation, BPAECs were treated
with either H2O2 (100 µM), vanadate (10 µM), or H2O2 (100 µM) plus vanadate (10 µM), and changes in albumin clearance were measured.
H2O2 (100 µM) altered albumin clearance from
70 ± 15 (control) to 240 ± 34 nl/min after a 2-h challenge, representing a threefold increase in albumin flux. Interestingly, pretreatment of cells with vanadate (10 µM) had no effect on albumin clearance; however, it potentiated the albumin clearance induced by
H2O2 from 240 to 580 nl/min (data not shown).
The effect of vanadate on H2O2-induced
permeability change is consistent with the notion that vanadate
not only acts as an inhibitor of phosphatases but reacts with
H2O2 to generate peroxovanadium compounds like DPV (37, 40). Peroxovanadium compounds exhibit dual
properties as potent activators of TyKs and inhibitors of PTPases
(32, 37). We therefore investigated the effect of DPV on
EC barrier function. DPV increased EB-albumin clearance (Fig.
1A) and decreased electrical
resistance across EC monolayers (normalized resistance after 3 h
of treatment: vehicle, 0.94 ± 0.08; 1 µM DPV, 1.36 ± 0.12; 5 µM DPV, 0.62 ± 0.09; 10 µM DPV, 0.40 ± 0.05) in a dose-dependent fashion (Fig. 1B).
DPV-induced decreases in electrical resistance dropped below basal
values 60 min after addition of DPV, which was consistently preceded by
increases in electrical resistance. This barrier enhancement lasted for
15-30 min post-DPV challenge and was followed by substantial
decreases in electrical resistance 2 h after addition of DPV. To
exclude cytotoxicity as the mechanism of DPV-mediated barrier
dysfunction, we determined 2-[3H]deoxyglucose release
(33) after challenge with H2O2,
vanadate, H2O2 plus vanadate, and DPV. The
cytotoxic index, expressed as percent of control value, ranged from 2.8 to 9.9% with exposure to various ROS, indicating minimal cytotoxicity
(data not shown).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Diperoxovanadate (DPV) induces endothelial cell (EC)
barrier dysfunction. Bovine pulmonary artery ECs (BPAECs) grown on
polycarbonate membranes with complete medium 199 (A) or on
gold microelectrodes (B) were pretreated for 1 h with
medium 199 followed by addition of indicated concentrations of DPV.
A: clearance of albumin coupled to Evans blue dye across
cell monolayers was determined for 2 h. Values are means ± SE of 3 independent experiments. Significantly different from vehicle:
* P < 0.05; ** P < 0.005. B: measurement of transendothelial electrical resistance was
carried out as described in METHODS. Tracings are
representative of 3 independent experiments.
|
|
TyK inhibition attenuated DPV-induced barrier dysfunction.
Because DPV modulated TyK and PTPase activities in ECs
(33), we examined the effect of TyK inhibitors on
DPV-induced protein tyrosine phosphorylation and EC barrier function.
Genistein (100 µM), an inhibitor of both receptor and non-receptor
TyKs, partially attenuated both basal and DPV-induced albumin clearance
and electrical resistance (Fig. 2).
Interestingly, herbimycin, which is known to block Src kinases
(21), also partially blocked the DPV-mediated electrical
resistance seen after 2 h (Fig. 2B). We next examined the effect of PP-2, a more specific inhibitor of the Src family of
non-receptor TyKs (13), on DPV-induced barrier
dysfunction. PP-2 (1 µM) attenuated DPV-induced albumin clearance (by
~50%) but had no significant effect on basal albumin clearance (Fig. 3). Similarly, PP-2 attenuated
DPV-induced EC electrical resistance in a dose- and time-dependent
manner (Table 1). In independent experiments, no change in EC morphology or cytotoxicity was observed after PP-2 (1-50 µM) treatment (data not shown). These data
strongly suggest the involvement of the Src family of non-receptor
TyKs in DPV-induced EC barrier dysfunction.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 2.
Tyrosine kinase inhibition attenuates DPV-induced EC
barrier dysfunction. A: BPAECs grown on polycarbonate
membranes were pretreated with genistein (Gen; 100 µM) for 1 h
followed by treatment with DPV (5 µM). Clearance of Evans blue
dye-albumin across the monolayers was measured for 2 h at 10-min
intervals. Values are means ± SE of 3 independent experiments.
* Significantly different from vehicle, P < 0.01. ** Significantly different from DPV, P < 0.05. B: BPAECs grown on gold microelectrodes were pretreated with
medium 199, Gen, or herbimycin (Herb) for 1 h before addition of
DPV. Changes in electrical resistance were monitored for the next 180 min as described in METHODS. Tracings are representative of
3 independent experiments.
|
|

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 3.
Src inhibition with PP-2 attenuates DPV-induced EC
barrier dysfunction. BPAECs grown on polycarbonate membranes were
pretreated with medium 199 (vehicle) or PP-2 (1 µM) for 1 h
before treatment with DPV (5 µM). Clearance of Evans blue dye-albumin
across cell monolayers was determined for 2 h. Values are
means ± SE of 3 independent experiments.* Significantly
different from vehicle, P < 0.05. ** Significantly
different from DPV, P < 0.01.
|
|
DPV increased Src activation.
DPV (5 µM) enhanced protein tyrosine phosphorylation in a
time-dependent manner as evidenced by phosphotyrosine
immunofluorescence and Western blot analysis of total EC lysates
(12, 34). To further study the effect of DPV on activation
of specific TyKs, BPAECs were challenged with vehicle or DPV (5 µM)
for varying time periods; Src immunoprecipitates from control or
DPV-treated cell lysates were subjected to SDS-PAGE, and Src activation
was determined by tyrosine phosphorylation, autophosphorylation with [
-32P]ATP, or phosphorylation of raytide peptide
substrate. Increased Src autophosphorylation was observed as early as 2 min after DPV treatment and returned to near basal level by 30 min,
whereas increased Src protein tyrosine phosphorylation was sustained
beyond 30 min (Fig. 4).
Densitometric analysis of the pooled data from three experiments showed
2.6- and 10-fold increases in Src autophosphorylation at 5 and 30 min,
respectively, after DPV treatment (Table
2). Interestingly, an increase in Src
autophosphorylation and raytide peptide phosphorylation was observed as
early as 2 min of DPV treatment (Table 2). SDS-PAGE of Src
immunoprecipitates followed by Western blotting with Src antibody
indicated almost equal loading on the gels (Fig. 4). These results show
that DPV rapidly increases Src activity and tyrosine phosphorylation.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 4.
Activation of Src kinase by DPV. Confluent BPAECs (60-mm
dishes) were treated with DPV for indicated time periods, and cell
lysates were immunoprecipitated with Src antibodies. Src
immunoprecipitates (IP) were subjected to SDS-PAGE and probed with
anti-Src antibody or Src autophosphorylation activity as described in
METHODS. A: Western blot analysis with
anti-phosphotyrosine (PTyr) antibody. B: autophosphorylation
(Autophos) with [ -32P]ATP. C: Western
blotting with anti-Src antibody. Blots are representative of 3 independent experiments. IB, immunoblot.
|
|
We next determined whether the barrier protective effect elicited by
PP-2 was linked to modulation of Src kinase activation. PP-2 (1-50
µM) attenuated DPV-induced increases in protein tyrosine phosphorylation of total EC proteins (Fig.
5A), tyrosine phosphorylation of Src and Src autophosphorylation (Fig. 5B). Densitometric
analysis of the data from three independent experiments showed that
PP-2 (1 µM) treatment substantially and significantly attenuated the DPV-induced Src activation (Fig. 5C). These results are
consistent with a mechanistic link between DPV-induced Src activation
and EC barrier dysfunction.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
PP-2 attenuates DPV-induced Src activation in ECs. Confluent BPAECs
in 100-mm dishes were pretreated with MEM or MEM containing varying
concentrations of PP-2 for 1 h followed by treatment with (+) and
without ( ) DPV for 30 min. Cell lysates were prepared under native
conditions with modified radioimmunoprecipitation assay buffer for
electrophoresis and immunoprecipitation. A: cell lysates (5 µg of protein) were subjected to electrophoresis on 10%
polyacrylamide gels, transferred to nitrocellulose membranes, and
immunoblotted with anti-PTyr antibody. Nos. at left,
molecular mass. B: Src immunoprecipitates were subjected to
Western blotting with anti-PTyr antibody (top),
autophosphorylation with [ -32P]ATP
(middle), and Western blotting with anti Src antibody
(bottom) as described in METHODS. C:
Src immunoprecipitates from control or DPV-treated ECs were subjected
to Western blotting for Src tyrosine phosphorylation, Src
autophosphorylation, and raytide peptide phosphorylation as described
above. Densitometric analyses of the blots were performed with a
Bio-Rad densitometer. Data are averages of 3 independent experiments.
|
|
To further determine the specificity of PP-2 on DPV-mediated EC barrier
dysfunction, we investigated the effect of PP-2 on DPV- and phorbol
12-myristate 13-acetate (PMA)-induced decreases in electrical
resistance. Treatment of BPAECs with DPV (5 µM) or PMA (100 nM) for
varying time periods (60-180 min) decreased electrical resistance
(Fig. 6). Pretreatment of cells with PP-2 (1 µM) had no effect on the PMA-mediated decrease in electrical resistance; however, the DPV-induced EC barrier dysfunction was partially attenuated (normalized resistance after 3 h of
treatment: vehicle, 1.14 ± 0.09; 5 µM DPV, 0.72 ± 0.12;
100 nM PMA, 0.77 ± 0.04; 1 µM PP-2, 0.94 ± 0.06; 1 µM
PP-2 plus 5 µM DPV, 1.06 ± 0.14; 1 µM PP-2 plus 100 nM PMA,
0.64 ± 0.07). Similarly, PP-2 (50 µM) showed no effect on
PMA-mediated barrier dysfunction (data not shown). Under similar
experimental conditions, the PMA-induced electrical resistance was
abolished by pretreating cells with a known PKC inhibitor,
bisindolylmaleimide (data not shown). These results suggest
that the protective effect of PP-2 on EC barrier dysfunction is agonist
specific.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 6.
Src inhibition does not attenuate phorbol 12-myristate
13-acetate (PMA)-induced EC barrier dysfunction. Confluent BPAECs grown
on gold microelectrodes were pretreated with medium 199 or medium 199 containing PP-2 for 1 h followed by the addition of DPV or PMA.
Changes in electrical resistance were determined for an additional
3 h as described in METHODS. Tracings are
representative of 3 independent experiments.
|
|
Src overexpression altered DPV-induced EC barrier dysfunction.
We next examined DPV-induced changes in electrical resistance in ECs
transiently overexpressing either a wild-type Src construct, a
constitutively active Src mutant, or a dominant negative Src mutant and
compared the results to empty vector-transfected cells. The Src
expression level as determined by Western blotting compared with vector
alone or endogenous p60Src indicated a
significant increase in expression (Fig.
7A). Interestingly, as
indicated by the manufacturer (Upstate Biotechnology), in the transfected cells, the expressed Src protein exhibited a retarded mobility on SDS-PAGE compared with native p60Src
(Fig. 7A). The kinase activity present in Src
immunoprecipitates from cells transfected with the dominant negative
Src mutant was much lower than the activity present in the wild-type
Src or the constitutively active mutant of Src-transfected ECs (Fig.
7A). Also, DPV-induced tyrosine phosphorylation of Src was
lower in ECs expressing dominant negative Src mutant, whereas wild-type Src and constitutively active Src increased DPV-mediated tyrosine phosphorylation (data not shown). Next, we examined the effects of
transient expression of wild-type Src, constitutively active Src, and
dominant negative Src on DPV-induced changes in EC electrical resistance. As shown in Fig. 7B, expression of dominant
negative Src attenuated DPV-induced electrical resistance compared with vector alone-transfected cells, whereas monolayers overexpressing wild-type Src or constitutively active Src exhibited a higher decrease
in electrical resistance compared with basal or DPV treatment (normalized resistance after 3 h: vector, 1.17 ± 0.14; 5 µM DPV, 0.63 ± 0.13; wild-type Src, 0.95 ± 0.08;
wild-type Src plus 5 µM DPV, 0.51 ± 0.07; constitutively active
Src, 0.99 ± 0.12; constitutively active Src plus 5 µM DPV,
0.38 ± 0.02; dominant negative Src, 0.93 ± 0.06; dominant
negative Src plus 5 µM DPV, 0.80 ± 0.08). These results provide
strong evidence that Src activation is an important regulatory event in
DPV-induced EC barrier dysfunction.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 7.
Src expression alters DPV-induced EC barrier dysfunction.
A: confluent BPAECs in 100-mm dishes were transfected with
either empty vector or wild-type (wt) Src, constitutively active (+)
Src, or dominant negative ( ) Src cDNAs with FuGENE 6 as described in
METHODS. After transfection, Src was immunoprecipitated
from the cell lysates with a polyclonal anti-Src antibody under native
conditions. Src immunoprecipitates were subjected to electrophoresis in
10% polyacrylamide gels, transferred to nitrocellulose membranes, and
immunoblotted with anti-Src or anti-PTyr antibodies. Src activity in
the immunoprecipitates was measured with [ -32P]ATP and
raytide peptide as a substrate as described in METHODS.
Nos. at left, molecular mass. B: BPAECs grown on
gold microelectrodes were transfected with empty vector or wild-type
Src, constitutively active Src, or dominant negative Src mutants. After
transfection (48 h), cells were treated with medium 199 or medium 199 containing DPV for 4 h, and changes in electrical resistance were
measured as described in METHODS. Tracings are
representative of 3 independent experiments.
|
|
DPV enhances association of contractile and adherens junction
proteins in Src immunoprecipitates.
The Src family of non-receptor kinases are localized in focal plaques
and mediate phosphorylation of focal adhesion kinases (FAKs),
p130Cas, paxillin, and MLCK in response to an
external stimulus (12, 19). To further define the
potential regulation of DPV-induced EC barrier dysfunction by Src, we
investigated the possible association of contractile and adherens
junction proteins in Src immunoprecipitates before and after DPV
challenge. Western blot analysis of Src immunoprecipitates obtained
from control and DPV-treated cells under nondenaturing conditions
revealed a marked increase in tyrosine-phosphorylated proteins
(60-214 kDa) associated with Src (Fig.
8). Two proteins subsequently identified
by Western blotting in the Src immunoprecipitates were the novel high
molecular weight 214-kDa MLCK (53) and the p80/85
actin-binding protein cortactin (Fig. 8). Immunoreactive MLCK was not
associated with Src immunoprecipitates prepared from control cells
prepared under native conditions. However, after DPV challenge, there
was a time-dependent increase in MLCK associated with Src. In contrast
to MLCK, there was a significant association of p80 and p85 cortactin
isoforms with Src even under basal conditions, and stimulation with DPV
(5 µM) further enhanced the level of association (Fig. 8). These
findings suggest that DPV treatment increases the association of MLCK
and cortactin with Src and that MLCK and cortactin may represent
important downstream targets for Src in regulating EC barrier function.

View larger version (39K):
[in this window]
[in a new window]
|
Fig. 8.
DPV enhances the stable association of myosin light chain
kinase (MLCK) and cortactin in Src immunoprecipitates. Confluent BPAECs
in 100-mm dishes were treated with MEM or MEM containing DPV (5 µM)
for indicated time periods. Cells were washed in ice-cold PBS, and cell
lysates were prepared in lysis buffer as described in
METHODS. Cell lysates (1 mg of protein) were subjected to
immunoprecipitation with anti-Src antibody, and immunoprecipitates were
subjected to electrophoresis on 10% polyacrylamide gels, transferred
to nitrocellulose membranes, and immunoblotted with anti-PTyr antibody
(A), anti-MLCK antibody (B), and anti-cortactin
antibody (C). Blots are representative of 3 independent
experiments. Nos. at left, molecular mass.
|
|
 |
DISCUSSION |
The structural and functional integrity of the vascular
endothelium is critical to normal lung function and vessel wall
homeostasis. Injury to the endothelium results in impaired barrier
function, with exudation of fluids and proteins into the interstitium
and alveoli (29). ROS released from activated
polymorphonuclear leukocytes induced changes in intracellular levels of
Ca2+ (39), thiols (43), and
high-energy nucleotides (55), resulting in cell injury and
dysfunction. Although the role of ROS in EC cell injury has been well
studied with cells in culture and in ischemia-reperfusion systems
(24, 54), the mechanism(s) of ROS-induced EC barrier
dysfunction has yet to be completely defined. Earlier studies
(10, 27) with macro- and microvascular ECs suggested that
rapid activation of phosphatidylinositol 4,5-bisphosphate-specific phospholipase (PL) C and generation of diacylglycerol and inositol 1,4,5-trisphosphate second messengers may be involved in thrombin- and
H2O2-induced increases in permeability.
Exposure of dermal microvascular ECs to PMA (30, 41) or
H2O2 (45) increased albumin flux
across the monolayer in association with activation and translocation
of PKC-
to the membrane (45). However, the protein
target(s) involved in the regulation of ROS-induced EC barrier
dysfunction is not known. Interestingly,
-thrombin- or PMA-mediated
activation of PKC enhanced phosphorylation of the actin-, myosin-, and
calmodulin-binding protein caldesmon and the intermediate filament
vimentin and also enhanced albumin permeability across EC monolayers,
suggesting a role for cytoskeletal proteins in EC barrier dysfunction
(47). A similar role for PKC in the thrombin-induced
increase in transendothelial permeability to albumin, which was
attenuated by PKC inhibitors (10, 27), was also observed.
Pretreatment of guinea pig lungs with H-7, a PKC inhibitor, prevented
pulmonary edema in response to perfusion with
H2O2, suggesting involvement of PKC in
permeability changes (20). Similarly, changes in
intracellular Ca2+, mediated by inositol
1,4,5-trisphosphate or by other mechanism(s), may also contribute to
the EC barrier dysfunction observed with agonists or ROS
(39).
The results presented in this study demonstrate for the first time a
role for Src kinases in DPV-induced EC barrier dysfunction. An increase
in permeability to albumin or a decrease in electrical resistance
induced by DPV, a potent inhibitor of PTPases and activator of TyKs
(32, 37), was attenuated by genistein, suggesting a role
for protein tyrosine phosphorylation. DPV-induced reduction in
electrical resistance was always preceded by a barrier enhancement that
lasted for 15-30 min. (Fig. 1B). At this time, it is
unclear what the significance of this initial increase in resistance to barrier function is, but it may represent an early barrier protective mechanism. DPV rapidly activated Src kinase as evidenced by increased autophosphorylation and raytide peptide phosphorylation, and changes in
EC barrier dysfunction were seen 2 h later with Src activation. Pretreatment of ECs with PP-2, a recently described specific inhibitor of the Src family of non-receptor kinases (13), attenuated
DPV-induced Src activation and increased permeability to albumin,
strongly suggesting a role for Src in barrier dysfunction. The barrier protective effect of PP-2 was specific toward DPV-mediated Src kinase
activation and barrier dysfunction because PP-2 had no effect on
PMA-mediated decrease in electrical resistance. The present study
employing a pharmacological Src kinase inhibitor was complemented by
additional investigations with overexpression of wild-type Src,
constitutive active Src, and dominant negative Src plasmids, which also
significantly altered DPV-induced EC electrical resistance. Our results
also show that transient expression of constitutively active Src is not
sufficient to alter basal electrical resistance. It is possible that an
additional signaling pathway(s), such as changes in intracellular
Ca2+, may be necessary to modulate DPV-induced barrier
function. An earlier study (32) has demonstrated that DPV
increased intracellular Ca2+ in ECs that was attenuated by
chelators of Ca2+. Further experiments to demonstrate the
role for Ca2+ in DPV-mediated EC barrier dysfunction by Src
are needed.
TyKs and, in particular, the Src family of non-receptor TyKs play an
important role in transducing signals from cell exterior to cell
interior. In response to growth factors, oxidative and shear stress,
ultraviolet light, and a variety of agonists including thrombin and
angiotensin II, Src is activated, as evidenced by enhanced specific
activity and increased protein tyrosine phosphorylation (35,
51). In our studies, autophosphorylation and tyrosine phosphorylation of Src were detected as early as 2 min after DPV, which
was similar but not identical to that observed with thrombin stimulation of lung fibroblasts (4) and angiotensin II
activation of p60Src in vascular smooth muscle
cells (18). Although regulation of Src kinase family
members involves phosphorylation of tyrosine-416 and dephosphorylation
of tyrosine-527 (48), it is unclear which of these two
tyrosine residues is involved in DPV-induced Src activation. However,
because DPV is known to inhibit PTPase activity and/or to activate TyKs
(34), it is reasonable to assume that the DPV-induced
activation of Src involves either of the pathways. It is also known
that additional phosphorylation sites, including phosphorylation of
serine-12 of Src, may have functional consequences on kinase activity
(48), and analyses of the phosphorylation sites on Src in
control and DPV-challenged ECs should provide further insight into the
mechanism of its activation under oxidative stress.
Our finding that DPV stimulates Src and that this event represents an
important mechanism for EC barrier dysfunction is consistent with a
previous report (42) of the possible involvement of Src in
thrombin-induced EC permeability changes and electrical resistance. Because paracellular transport of macromolecules across the monolayer is regulated by contractile and tethering forces (11),
activation of Src kinase by DPV may involve tyrosine phosphorylation of
other downstream target proteins such as adherens junction proteins, FAKs, and actomyosin contractile proteins regulating barrier function. One potential key cytoskeletal target is the EC MLCK, the activity of
which is a major determinant of tension development. EC MLCK activity
is regulated by Ser/Thr phosphorylation (53), and Garcia et al. (12) and Shi et al. (42) have
recently demonstrated a novel regulation of the enzyme by tyrosine
phosphorylation. A recent study (12) indicated that DPV
evokes significant endothelial contraction, tyrosine phosphorylation of
MLCK, and MLCK activation in BPAECs (12). The DPV-induced
MLC phosphorylation and EC contraction were attenuated by either C3
exotoxin from Clostridium botulinum or MLCK inhibitors,
consistent with dual mechanisms that regulate the level of MLC
phosphorylation in ECs involving Rho GTPase-mediated inhibition of MLC
phosphatase and regulation of MLCK activity via tyrosine
phosphorylation (12). The results reported here on the
association of MLCK in Src immunoprecipitates after DPV treatment
implicate Src as the effector kinase in catalyzing phosphorylation of
tyrosine residues in MLCK. A recent study (53) on the
cloning of the 214-kDa EC MLCK indicated that this unique isoform is
present predominantly in nonmuscle tissue such as ECs compared with the 130- to 160-kDa isoform in smooth muscle cells (53). This
unique MLCK from ECs contains consensus sequences for Src kinases, PKC, and the calmodulin-protein kinase II region in the novel
NH2 terminus that are not present in the smooth muscle
isoform (23). Enhanced tyrosine phosphorylation of MLCK
may result in increased protein-protein interaction involving Src
homology (SH) 2 and SH3 domains that have also been identified in the
EC MLCK isoform (Garcia, unpublished data). Also, the presence
of the SH3 motif could support its role as a scaffolding and adaptor
protein. We also identified an association of the p80/85 actin-binding
protein cortactin in Src immunoprecipitates. Cortactin is tyrosine
phosphorylated by Src family of non-receptor TyKs in response to
external stimuli (25). Although the physiological role of
tyrosine phosphorylation of cortactin is not well understood, a recent
study (17) indicated that cortactin cross-links
filamentous actin in vitro that is downregulated by Src-dependent
tyrosine phosphorylation. Src-mediated tyrosine phosphorylation of
cortactin resulted in an enhanced motility of EC304 ECs, suggesting a
possible role in angiogenesis (16). It is possible that
DPV-mediated phosphorylation of cortactin by Src in ECs may be involved
in cytoskeletal reorganization and barrier dysfunction. Further studies on the sites of tyrosine phosphorylation in cortactin by DPV and sites
of interaction between cortactin and other cytoskeletal proteins would
give a better understanding of the role of cortactin in EC barrier function.
In addition to Src, H2O2 and DPV stimulate the
tyrosine phosphorylation of p125FAK, paxillin, caveolin,
and mitogen-activated protein kinases in vascular ECs and smooth muscle
cells (1, 52). Because rearrangement of cytoskeletal
proteins and focal adhesion proteins plays an important role in
determining EC shape, migration, proliferation, and barrier function,
phosphorylation-dephosphorylation of focal adhesion proteins may have a
role in barrier function. A recent study (18) in vascular
smooth muscle cells suggested that thrombin and angiotensin II cause
actin stress fibers and focal adhesion protein assembly through Src
activation and increased phosphorylation of Cas, paxillin, and tensin
(18). The mechanism(s) by which the FAK-associated
signaling complex alters the actin cytoskeleton and barrier dysfunction
is unclear but may involve small G proteins, other cytoskeletal
proteins, and focal adhesion proteins. A recent study by Vepa et al.
(52) showed that H2O2 and DPV
stimulate tyrosine phosphorylation of FAK and paxillin in BPAECs and
that DPV is a potent stimulator of p42/p44 mitogen-activated protein kinases, consistent with this notion. Interestingly, we have noted that
although the DPV-induced EC barrier dysfunction was inhibited by both
PP-2 and genistein, neither pharmacological agent reduced DPV-induced
tyrosine phosphorylation of FAK or paxillin (Vepa S and Natarajan V,
unpublished data), suggesting either that these effectors may not
directly participate in EC barrier dysfunction or that spatial location
of these proteins may not be linked to their phosphorylation status.
The present studies do not exclude other signaling pathways such as
mitogen-activated protein kinases, phospholipases, and receptor TyKs in
DPV-mediated EC barrier dysfunction. Preliminary studies suggest that
DPV-induced phospholipase D (PLD) activation with subsequent generation
of phosphatidic acid increases the permeability to albumin across EC
monolayers (Natarajan and Shi, unpublished observations). The mechanism
of induction of endothelial monolayer permeability by phosphatidic acid
is not known. Phosphatidic acid is recognized as a second messenger and
can phosphorylate intracellular proteins through activation of
phosphatidic acid-dependent protein kinases. Another pathway that may
be modulated by DPV and other peroxovanadium compounds is altering the
cellular thiol redox status. Because peroxovanadium compounds oxidize
cysteine residues of protein tyrosine phosphatases, it is conceivable
that DPV also alters cysteine residues in Src and other signaling
proteins regulating barrier function. Further studies on phosphatidic
acid-dependent activation of protein kinases and redox regulation of
Src and PLD activation should provide a better understanding of other mechanisms involved in endothelial barrier dysfunction.
In summary, the data presented here demonstrate that DPV-induced
changes in EC permeability are regulated by protein tyrosine phosphorylation involving activation of Src kinase as an early and
important upstream signaling mechanism that regulates EC barrier dysfunction. Our data also indicate that association and tyrosine phosphorylation of cortactin and MLCK by Src may represent downstream signaling pathways regulating DPV-induced EC permeability. A recent study (50) suggested the feasibility of suppression of Src
activity for gene therapy in rheumatoid arthritis, and our studies also suggest that modulation of Src activity by specific Src inhibitors or
dominant negative Src could represent a viable therapeutic treatment
for pulmonary edema and endothelial dysfunction. A proposed model of
DPV- or ROS-induced barrier dysfunction involving Src, cortactin, and MLCK is illustrated in Fig.
9.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 9.
Model for reactive oxygen species-induced regulation of EC barrier
function by Src, MLCK, and cortactin. Tyr-P, tyrosine phosphorylation;
P-Tyr, phosphorylated tyrosine; MLC-P, myosin light chain
phosphorylation; PPase, protein phosphatase.
|
|
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Drs. S. Vepa, K. Schaphorst, and A. Verin
for helpful discussions, Patricia Lyon for typing the manuscript, and
Lakshmi Natarajan for technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grants HL-57260, HL-58064, and HL-50533.
Address for reprint requests and other correspondence: V. Natarajan, Johns Hopkins Asthma and Allergy Bldg., Division of
Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Circle,
Baltimore, MD 21224 (E-mail: vnataraj{at}welch.jhu.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 24 January 2000; accepted in final form 11 April 2000.
 |
REFERENCES |
1.
Baas, AS,
and
Berk BC.
Differential activation of mitogen-activated protein kinases by H2O2 and O
2 in vascular smooth muscle cells.
Circ Res
77:
29-36,
1995[Abstract/Free Full Text].
2.
Bernard, GR,
Artigas A,
and
Brigham KL.
The American-European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination.
Am J Respir Crit Care Med
149:
818-824,
1994[Abstract].
3.
Brigham, KL.
Role of free radicals in lung injury.
Chest
89:
859-863,
1986[Abstract].
4.
Chen, Y-H,
Pouyssegur J,
Courtneidge SA,
and
Van Obberghen-Schilling E.
Activation of Src family kinase activity by the G-protein-coupled thrombin receptor in growth responsive fibroblasts.
J Biol Chem
269:
27372-27377,
1994[Abstract/Free Full Text].
5.
Eckenhoff, RG,
Dodia C,
Tan Z,
and
Fisher AB.
Oxygen-dependent reperfusion injury in isolated rat lung.
J Appl Physiol
72:
1454-1460,
1992[Abstract/Free Full Text].
6.
Fajardo, LF.
The complexity of endothelial cells.
Am J Clin Pathol
92:
241-250,
1989[ISI][Medline].
7.
Finkel, T.
Oxygen radicals and signaling.
Curr Opin Cell Biol
10:
248-253,
1998[ISI][Medline].
8.
Fishman, AP.
Endothelium: a distributed organ of diverse capabilities.
Ann NY Acad Sci
401:
1-8,
1982[ISI][Medline].
9.
Fishman, AP,
and
Pietra GG.
Handling of vasoactive materials by the lung.
N Engl J Med
291:
884-890,
1974[ISI][Medline].
10.
Garcia, JGN,
Pavalko FM,
and
Patterson CE.
Vascular endothelial cell activation and permeability responses to thrombin.
Blood Coagul Fibrinolysis
6:
609-626,
1995[ISI][Medline].
11.
Garcia, JGN,
and
Schaphorst KL.
Regulation of endothelial gap formation and paracellular permeability.
J Investig Med
43:
117-126,
1995[ISI][Medline].
12.
Garcia, JGN,
Verin AD,
Schaphorst KL,
Siddiqui R,
Patterson CE,
Csortos C,
and
Natarajan V.
Regulation of endothelial cell myosin light chain kinase by Rho, cortactin, and p60src.
Am J Physiol Lung Cell Mol Physiol
276:
L989-L998,
1999[Abstract/Free Full Text].
13.
Hanke, JH,
Gardner JP,
Dow RL,
Changelian PS,
Brissette WH,
Weringer EJ,
Pollok BA,
and
Connelly PA.
Discovery of a novel, potent and Src family selective tyrosine kinase inhibitor. Study of Lck- and Fyn-dependent T cell activation.
J Biol Chem
271:
695-701,
1996[Abstract/Free Full Text].
14.
Harlan, JM.
Leukocyte-endothelial interactions.
Blood
65:
513-525,
1985[ISI][Medline].
15.
Hogg, JC,
and
Doershuck CM.
Leukocyte traffic in the lung.
Annu Rev Physiol
57:
97-114,
1995[ISI][Medline].
16.
Huang, C,
Liu J,
Haudenschild CC,
and
Zhan X.
The role of tyrosine phosphorylation cortactin in the locomotion of endothelial cells.
J Biol Chem
273:
25770-27556,
1998[Abstract/Free Full Text].
17.
Huang, C,
Ni Y,
Wang T,
Gao Y,
Haudenschild CC,
and
Zhan X.
Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation.
J Biol Chem
272:
13911-13915,
1997[Abstract/Free Full Text].
18.
Ishida, M,
Marrero MB,
Schieffer B,
Ishida T,
Bernstein KE,
and
Berk BC.
Angiotensin II activates pp60c-Src in vascular smooth muscle cells.
Circ Res
77:
1053-1059,
1995[Abstract/Free Full Text].
19.
Ishida, T,
Ishida M,
Suero J,
Takahashi M,
and
Berk BC.
Agonist-stimulated cytoskeletal reorganization and signal transduction at focal adhesions in vascular smooth muscle cells require c-Src.
J Clin Invest
103:
789-797,
1999[Abstract/Free Full Text].
20.
Johnson, A,
Phillips P,
Hocking D,
Tsars M-F,
and
Ferro T.
Protein kinase inhibitor prevents pulmonary edema in response to H2O2.
Am J Physiol Heart Circ Physiol
256:
H1012-H1022,
1989[Abstract/Free Full Text].
21.
June, CH,
Flether MC,
Ledbetter JA,
Schieven GL,
Siegel JN,
Phillips AF,
and
Samelson LE.
Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction.
Proc Natl Acad Sci USA
87:
7722-7726,
1990[Abstract].
22.
Konishi, H,
Tanaka M,
Takemura Y,
Matsuzaki H,
Ono Y,
Kikkawa U,
and
Nishizuka Y.
Activation of protein kinase C by tyrosine phosphorylation in response to hydrogen peroxide.
Proc Natl Acad Sci USA
94:
11233-11237,
1997[Abstract/Free Full Text].
23.
Lazar, V,
and
Garcia JGN
A single human myosin light chain kinase gene (MLCK; MYLK) transcribes multiple nonmuscle isoforms.
Genomics
57:
256-267,
1999[ISI][Medline].
24.
Lefer, AM,
Tsao PS,
Lefer DJ,
and
Ma XL.
Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia.
FASEB J
5:
2029-2034,
1991[Abstract/Free Full Text].
25.
Levy-Toledano, S.
Platelet signal transduction pathways: could we organize them into a "hierarchy"?
Haemostasis
29:
4-15,
1999[ISI][Medline].
26.
Lopez-Ongil, S,
Torrecillas G,
Perez-Sala D,
Gonzalez-Santiago L,
Rodriguez-Puyol M,
and
Rodriguez-Puyol D.
Mechanisms involved in the contraction of endothelial cells by hydrogen peroxide.
Free Radic Biol Med
26:
501-510,
1999[ISI][Medline].
27.
Lum, H,
and
Malik AB.
Regulation of vascular endothelial barrier function.
Am J Physiol Lung Cell Mol Physiol
267:
L223-L241,
1994[Abstract/Free Full Text].
28.
Marchisio, PC,
D'Urso N,
Comoglio PM,
Giancotti FG,
and
Tarone G.
Vanadate-treated baby kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus transformed counterparts.
J Cell Biochem
37:
151-159,
1988[ISI][Medline].
29.
Marini, JJ,
and
Evans TW.
Acute Lung Injury. New York: Springer-Verlag, 1998.
30.
Nagpala, PG,
Malik AB,
Vuong PT,
and
Lum H.
PKC beta 1 overexpression augments phorbol ester-induced increase in endothelial permeability.
J Cell Physiol
166:
249-255,
1996[ISI][Medline].
31.
Natarajan, V.
Oxidants and signal transduction in vascular endothelium.
J Lab Clin Med
125:
26-37,
1995[ISI][Medline].
32.
Natarajan, V,
Scribner WM,
Al-Hassani M,
and
Vepa S.
Reactive oxygen species signaling through regulation of protein tyrosine phosphorylation in endothelial cells.
Environ Health Perspect
106, Suppl5:
1205-1212,
1998[ISI][Medline].
33.
Natarajan, V,
Taher MM,
Roehm B,
Parinandi NL,
Schmid HHO,
Kiss Z,
and
Garcia JGN
Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide.
J Biol Chem
268:
930-937,
1993[Abstract/Free Full Text].
34.
Natarajan, V,
Vepa S,
Shamlal R,
Al-Hassani M,
Ramasarma T,
Ravishanker HN,
and
Scribner WM.
Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate.
Mol Cell Biochem
183:
113-124,
1998[ISI][Medline].
35.
Parsons, JT,
and
Parsons SJ.
Src family of protein tyrosine kinases: cooperating with growth factor and adhesion signaling pathways.
Curr Opin Cell Biol
9:
187-192,
1997[ISI][Medline].
36.
Patterson, CE,
Rhoades RA,
and
Garcia JGN
Evans blue dye as a marker of albumin clearance in cultured endothelial monolayers and isolated lung.
J Appl Physiol
72:
865-873,
1992[Abstract/Free Full Text].
37.
Posner, BI,
Faure R,
Burgess JW,
Beven AP,
LaChance D,
Zhang-Sun G,
Fantug IG,
Ng JB,
Hall DA,
Lum BS,
and
Snaver A.
Peroxovanadium compounds. A new class of potent phosphotyrosine phosphatase inhibitors which are insulin mimetics.
J Biol Chem
269:
4596-4604,
1994[Abstract/Free Full Text].
38.
Ryan, JW,
and
Ryan US.
Pulmonary endothelial cells.
Fed Proc
36:
2683-2691,
1977[ISI][Medline].
39.
Schilling, WP,
and
Elliott SJ.
Ca2+ signaling mechanisms of vascular endothelial cells and their role in oxidant-induced endothelial cell dysfunction.
Am J Physiol Heart Circ Physiol
262:
H1617-H1630,
1992[Abstract].
40.
Shankar, HN,
and
Ramasarma T.
Multiple reactions in vanadyl-V (IV) oxidation by H2O2.
Mol Cell Biochem
129:
19-29,
1993.
41.
Shasby, DM,
Lind SE,
Shasby SS,
Goldsmith JC,
and
Hunninghake GW.
Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis.
Blood
65:
605-614,
1985[Abstract].
42.
Shi, S,
Verin AD,
Schaphorst KL,
Gilbert-McClain LL,
Patterson CE,
Irwin R,
Natarajan V,
and
Garcia JGN
Role of tyrosine phosphorylation in thrombin-induced endothelial cell contraction and barrier function.
Endothelium
6:
158-171,
1998.
43.
Sies, H.
Hydroperoxides and thiol oxidants in the study of oxidative stress in intact cells and organs.
In: Oxidative Stress, edited by Sies H.. Orlando, FL: Academic, 1985, p. 73-90.
44.
Siflinger-Birnboim, A,
Lum H,
Del Vecchio PJ,
and
Malik AB.
Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability.
Am J Physiol Lung Cell Mol Physiol
270:
L973-L978,
1996[Abstract/Free Full Text].
45.
Siflinger-Birnboim, A,
Schnitzer JE,
Del Vecchio PJ,
and
Malik AB.
Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability.
Lab Invest
67:
24-30,
1992[ISI][Medline].
46.
Staddon, JM,
Herrenknecht K,
Smales C,
and
Rubin LL.
Evidence that tyrosine phosphorylation may increase tight junction permeability.
J Cell Sci
108:
609-619,
1995[Abstract/Free Full Text].
47.
Stasek, JE,
Patterson CE,
and
Garcia JGN
Protein kinase C phosphorylates caldesmon 77 and vimentin and enhances albumin permeability across cultural bovine pulmonary artery endothelial cell monolayers.
J Cell Physiol
153:
62-75,
1992[ISI][Medline].
48.
Superti-Furga, G.
Regulation of the Src protein tyrosine kinases.
FEBS Lett
369:
62-66,
1995[ISI][Medline].
49.
Suzuki, YJ,
Forman HJ,
and
Sevanian A.
Oxidants as stimulators of signal transduction.
Free Radic Biol Med
22:
269-285,
1997[ISI][Medline].
50.
Takayanagi, H,
Juji T,
Miyazaki T,
Iizuka H,
Takahashi T,
Isshiki M,
Okada M,
Tanaka Y,
Koshihara Y,
Odla H,
Kurokawa T,
Nakamura K,
and
Tanaka S.
Suppression of arthritic bone destruction by adenovirus-mediated csk gene transfer to synoviocytes and osteoclasts.
J Clin Invest
104:
137-146,
1999[Abstract/Free Full Text].
51.
Thomas, SM,
and
Brugge JS.
Cellular functions regulated by Src family kinases.
Annu Rev Cell Dev Biol
13:
513-609,
1997[ISI][Medline].
52.
Vepa, S,
Scribner WM,
Parinandi NL,
English D,
Garcia JGN,
and
Natarajan V.
Hydrogen peroxide stimulates tyrosine phosphorylation of focal adhesion kinase in vascular endothelial cells.
Am J Physiol Lung Cell Mol Physiol
277:
L150-L158,
1999[Abstract/Free Full Text].
53.
Verin, AD,
Lazar V,
Torry RJ,
Labarrere CA,
Patterson CE,
and
Garcia JGN
Expression of a novel high molecular weight myosin light chain kinase in endothelium.
Am J Respir Cell Mol Biol
19:
758-766,
1998[Abstract/Free Full Text].
54.
Ward, PA.
Mechanisms of endothelial cell injury.
J Lab Clin Med
118:
421-426,
1991[ISI][Medline].
55.
Wilson, J,
Winter M,
and
Shasby DM.
Oxidants, ATP depletion and endothelial permeability to macromolecules.
Blood
76:
2578-2582,
1990[Abstract].
Am J Physiol Lung Cell Mol Physiol 279(3):L441-L451
1040-0605/00 $5.00
Copyright © 2000 the American Physiological Society