1 Department of Pharmacology, We investigated the hypothesis that cAMP-dependent protein
kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An
E1
endothelial permeability; adenovirus; cAMP-dependent protein kinase
INCREASES IN INTRACELLULAR levels of the second
messenger cAMP in endothelial cells are known to protect against
endothelial barrier dysfunction in response to proinflammatory
mediators, including histamine, thrombin, oxidants, and tumor necrosis
factor (5, 8, 22, 35, 40, 43, 48). This protective effect of cAMP has
been demonstrated in several experimental systems with use of in vitro
cell culture (8, 34, 40, 43, 48) and in intact organ models of barrier
function such as ischemia-reperfusion injury (2, 5, 6, 22, 35).
Associated with the cAMP-mediated improvement of barrier function,
endothelial cells become flattened (34), show decreased
interendothelial gap formation (21, 43), and exhibit tighter adhesion
to matrix and inhibition of migration (29). Furthermore, increased
endothelial permeability in response to hypoxia exposure, tumor
necrosis factor, or oxidant treatment was accompanied by decreased
intracellular cAMP levels (21, 27, 41). Thus substantial evidence
exists in support of the notion that increased cAMP levels in
endothelial cells promote endothelial barrier function.
The mechanisms by which cAMP functions to regulate endothelial
permeability are presumed to occur predominantly through activation of
the cAMP-dependent serine/threonine protein kinase (PKA). Studies of
PKA function have relied mostly on the use of cAMP analogs that
increase intracellular levels of cAMP, the use of reagents that
stimulate its production (i.e., forskolin), or inhibition of its
metabolism (i.e., phosphodiesterase inhibitors). In the absence of
cAMP, PKA is an inactive tetramer consisting of two regulatory and two
catalytic subunits. Binding of cAMP to the regulatory subunit lowers
the affinity for the catalytic subunits by four orders of magnitude,
promoting dissociation into a dimer of regulatory subunit and two
active monomers of the catalytic subunit (52).
The function of PKA has also been studied using pharmacological PKA
inhibitors such as Rp-cAMPS, a competitive inhibitor of cAMP for
binding the regulatory subunit of PKA (55), and isoquinoline-based inhibitors, which target the ATP-binding domain on the catalytic subunit (13). However, limited studies have been performed using these
PKA inhibitors to demonstrate a direct relationship between PKA
activation and the protection against mediator-induced endothelial barrier dysfunction (50). Recent work reported that forskolin (an
activator of adenylyl cyclase) inhibited voltage-sensitive Ca2+ channels in a
cAMP-independent manner in ventricular myocytes and PC-12 cells (4,
42), suggesting possible nonspecific effects of forskolin. Furthermore,
in bovine adrenal cells the inhibition of a
K+ current by cAMP analogs and
forskolin was not prevented by pharmacological inhibitors of PKA and
PKA inhibitor peptide (PKI) (14). These studies indicate that
cAMP-promoting agents such as forskolin, as well as cAMP, may have
PKA-independent activity and question whether the enhanced barrier
function associated with elevation of cAMP levels in endothelial cells
is entirely mediated through activation of PKA. Furthermore, full
activation of PKA appears to require phosphorylation of its catalytic
subunit by a phosphoinositide-dependent protein kinase (9, 10). This
latter observation raises the interesting possibility that PKA activity
is regulated by phosphorylation-dephosphorylation, which in turn
modulates the cell's responsiveness to cAMP. Therefore, physiological
activation of PKA is likely under multiple modulating signals.
The goal of the study is to test the hypothesis that PKA activation
protects against mediator-induced increases in endothelial permeability. The endogenous PKA inhibitor (PKI), highly selective for
PKA, has been isolated from rabbit muscle, and its primary amino acid
sequence has been identified (46, 56). This heat-stable 75-amino acid
protein has high affinity (0.2 nM) and specific binding for the peptide
substrate binding site on the catalytic subunit of PKA (52), providing
greater selective inhibition of PKA than pharmacological inhibitors.
For this study, a replication-deficient adenovirus (Ad) containing the
synthetic gene encoding the complete amino acid sequence of PKI (11)
was constructed and used for introduction of the
PKI gene into endothelial cells. The
Ad-mediated gene transfer method was preferred over other transfection
methods, since Ad vectors have been shown to have high transduction
efficiency in endothelial cells and do not require host cell
replication for gene expression (31).
The findings indicate that infection of endothelial cells with the
E1 Cell culture.
Human dermal microvascular endothelial cells (HMEC) (1) were maintained
in culture in MCDB 131 medium (GIBCO BRL, Gaithersburg, MD)
supplemented with 5% fetal bovine serum (FBS; HyClone, Logan, UT), 10 ng/ml human epidermal growth factor (Sigma Chemical, St. Louis, MO), 1 µg/ml hydrocortisone, and antibiotics. HMEC is an immortalized cell
line transformed by simian virus 40 (SV40) large T antigen and has been
shown to retain endothelial cell phenotypic and functional
characteristics. They exhibit the expected morphological and functional
endothelial phenotypes: they express and secrete von Willebrand's
factor, take up acetylated low-density lipoprotein, form tubes when
grown in Matrigel, and express CD31 (platelet endothelial cell adhesion
molecule-1), CD36, intercellular adhesion molecule-1, and CD44 (1).
They also bind purified T cells in a regulatable manner, and their
response to cytokines is comparable to that of nontransformed
endothelial cells. HMEC were passaged for 5-7 days when confluent
and used for studies at passages
25-40. The 293 cells were maintained in DMEM
supplemented with 10% FBS, 2 mM
L-glutamine, 100 µg/ml
penicillin, 100 U/ml streptomycin, and 10 mg/ml amphotericin B. All
cell cultures were maintained at 37°C in a humidified
CO2 incubator at 5%
CO2.
Ad-mediated expression of PKI gene.
The 251-bp DNA fragment encoding the complete amino acid sequence of
rabbit muscle PKI (11) was subcloned
into the shuttle vector pACCMV.pLpA, creating pACCMV-PKI. This vector
includes, in order, 0-1.3 map units from the left end of the Ad
type 5 (Ad5) genome and, in place of E1a and part of E1b sequences
(required for replication), the cytomegalovirus (CMV) immediate early
promoter, pUC19 polylinker, SV40 small T antigen intron and
polyadenylation signal sequences, and finally map units 9-17 of
Ad5 (19). Equimolar amounts of pACCMV-PKI (0.2 µg) were
cotransfected, by cationic liposomes (Lipofectamine, GIBCO/BRL, Grand
Island, NY), with the plasmid pJM17 (0.8 µg), which contains the
full-length Ad (Ad5) genome sequences (with a mutant E3 region), as
well as ampicillin and tetracycline resistance sequences and a
bacterial origin of replication (33), into 293 cells, a transformed
renal embryonal kidney cell line (CRL 1573, American Type Culture
Collection) (20). Homologous recombination between the two plasmids
resulted in an E1
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,
E3
, replication-deficient
adenovirus (Ad) vector was constructed containing the complete sequence
of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI
resulted in overexpression of PKI.
Treatment with 0.5 µM thrombin increased transendothelial albumin
clearance rate (0.012 ± 0.003 and 0.035 ± 0.005 µl/min for
control and thrombin, respectively); the increase was prevented with
forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment.
Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC
infected with ultraviolet-inactivated AdPKI, the F + I-induced
inhibition was present. Also, F + I treatment of HMEC transfected with
reporter plasmid containing the cAMP response element-directed
transcription of the luciferase gene resulted in an almost threefold
increase in luciferase activity. Overexpression of
PKI inhibited this induction of
luciferase activity. The results show that Ad-mediated overexpression
of PKI in endothelial cells abrogated
the cAMP-mediated protection against increased endothelial
permeability, providing direct evidence that cAMP-dependent protein
kinase promotes endothelial barrier function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,
E3
, replication-deficient
recombinant Ad vector is highly efficient (>95%), does not alter
baseline endothelial barrier function, and is not cytotoxic. Infection
of endothelial cells with AdPKI resulted in overexpression of
PKI, which abrogated the cAMP-mediated inhibition of the increased endothelial permeability in response to
thrombin. Overexpression of PKI also
inhibited the cAMP response element (CRE)-directed transcription of the
reporter luciferase gene. Thus the results provide direct evidence that
activation of the PKA pathway is critically important in conferring
protection against mediator-induced endothelial barrier dysfunction.
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
,
E3
Ad genome that can
replicate and be packaged into virions only in 293 cells in which E1
function is supplied in
trans by integrated, constitutively expressed Ad E1 sequences. The vector AdPKI was amplified in 293 cells, and its genome was confirmed by PCR
amplification of contiguous Ad/expression cassette sequences. The
vector was purified by double cesium chloride ultracentrifugation,
exhaustively dialyzed against virus suspension buffer (10 mM Tris, 10 mM MgCl2, 10% glycerol), titered,
and stored at
80°C.
gal was constructed by subcloning the Escherichia coli
lacZ gene sequences into pACCMV.pLpA shuttle vector.
Additional control vectors were made by inactivating Ad vectors with
use of heat (65°C overnight) or ultraviolet (UV) irradiation (254 nm for 60 min).
Northern blot analysis.
Northern blot analysis was performed to determine the level of
transcription of PKI in HMEC infected
with AdPKI. Total RNA was isolated from control noninfected HMEC or
HMEC infected for 24 h at 100 multiplicities of infection (MOI; equal
to plaque-forming units/target cell) of AdPKI or AdNull with use of the
RNA STAT-60 isolation kit (Tel-Test, Friendswood, TX). Equal amounts of
total RNA were loaded in 2.2 M formaldehyde-0.8% agarose gel,
electrophoresed, and transferred to nitrocellulose. The
32P-labeled PKI probe was
generated by use of the Prime-a-Gene Labeling Kit (Promega, Madison,
WI). The RNA blot was hybridized with the probe in hybridization
solution containing 50% formamide at 42°C overnight, and then it
was washed three times at room temperature with 0.1% SDS and 2×
saline-sodium citrate solution (Fisher Scientific, Pittsburgh, PA).
Equal loading of RNA samples was demonstrated by visualization of the
fluorescence of ethidium bromide bound to the 28S rRNA subunit. Kodak
X-Omat X-ray film was used to expose the blot at 80°C for
1-3 days.
-Galactosidase activity.
The efficiency of gene transfer into HMEC with use of the Ad vectors
was determined by microscopic evaluation of the fraction of HMEC
expressing
-galactosidase activity after infection with Ad
gal.
HMEC grown to confluency in six-well culture dishes were washed twice
in Ca2+- and
Mg2+-free PBS and fixed in 1.25%
PBS-glutaraldehyde solution for 5 min. The cells were washed twice in
Ca2+- and
Mg2+-free PBS and incubated for
4-6 h at 37°C in the X-gal staining solution (50 mM
Tris · HCl, pH 7.5, 2.5 mM ferriferrocyanide, 15 mM
NaCl, 1 mM MgCl2, 0.5 mg/ml
X-gal). Blue-stained HMEC were positive for
-galactosidase.
Luciferase assay. A reporter plasmid containing the firefly luciferase gene under the transcriptional control of multiple units of CRE (pADneo2-C6-BGL) was used as an index of the cAMP-dependent signaling pathway (24). HMEC were grown to 60-70% confluency in six-well culture dishes and incubated with pADneo2-C6-BGL in Lipofectamine at a ratio of 1:8 for 3 h at 37°C. The HMEC were washed and replaced with complete medium containing 10% FBS, and after incubation for 20 h the cells were treated with 10 µM forskolin + 1 µM 3-isobutyl-1-methylxanthine (F + I) overnight. The cells were washed twice with Ca2+- and Mg2+-free PBS and collected in reporter lysis reagent (Promega). Luciferase activity was determined from the cell extracts with use of the luciferase assay reagent system (Promega). Activity in relative light units was measured in a luminometer (model FB 12, Zylux, Maryville, TN) and reported as values normalized to protein (bicinchoninic acid kit; Pierce, Rockford, IL).
Transendothelial albumin clearance rate. The transendothelial albumin clearance rate across cultured monolayers of endothelial cells was determined using an in vitro system, as described previously (32). This system measures the diffusive flux of the tracer molecules across cell monolayers and consists of luminal and abluminal compartments separated by a polycarbonate microporous filter (0.8 µm pore diameter). Endothelial cells were seeded at 105 cells on each filter and grown for 3-4 days to attain confluency. Both compartments contained the same medium (DMEM, 20 mM HEPES, pH 7.4) at volumes of 700 µl and 25 ml, respectively, but only the luminal compartment contained Evans blue-labeled albumin tracer (0.67 mg/ml in 4% albumin) (44). The luminal compartment was fitted with a Styrofoam outer ring and floated in the abluminal medium so that fluid levels remained equal after repeated samplings from the abluminal compartment. The abluminal compartment was stirred continuously for rapid mixing, and the entire system was kept at 37°C by a thermostatically regulated water bath. Samples of 300 µl were removed from the abluminal compartment at 10-min intervals for 60 min, and optical density (OD) was read at 620 nm. The change in volume over time provided the clearance rate in microliters per minute, as determined by weighted least-squares nonlinear regression (BMDP Statistical Software, Berkeley, CA).
Cell viability assay.
The effects of Ad infection of HMEC on cell viability were determined
by reduction of the tetrazolium salt
3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium
bromide (MTT; Sigma Chemical) by mitochondrial dehydrogenases (25).
HMEC were plated at 50,000 cells/well in a 96-well tissue culture dish
and grown for 48 h. The cells were washed twice in Hanks' balanced
salt solution, infected with Ad vectors accordingly, and incubated with
0.5 mg/ml MTT for 3 h at 37°C. Subsequently, 0.04 M HCl in
-isopropanol was added, and the OD was read at 570 nm in a plate
reader. The OD units provided an index of enzymatic activity of living cells.
Statistics. Single sample data were analyzed by the two-tailed t-test; a multiple range (Scheffé's) test was used for comparisons of experimental groups with a single control group (49).
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RESULTS |
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Efficiency of Ad-mediated transgene expression.
HMEC were infected with Adgal at 10 or 100 MOI. As control, HMEC
were also infected with heat-inactivated Ad
gal at 100 MOI or AdNull
at 100 MOI. At 2 days postinfection with Ad
gal at 100 MOI, >95%
of HMEC were stained with X-gal, indicating a high level of
-galactosidase activity (Fig.
1A); at
10 MOI, <30% of HMEC were stained (Fig.
1B). HMEC infected with
heat-inactivated Ad
gal at 100 MOI (Fig.
1C) or AdNull at 100 MOI (Fig.
1D) did not stain with X-gal,
indicating lack of
-galactosidase activity. UV-irradiated Ad
gal
also showed inhibition of
-galactosidase activity (not shown).
|
Effects of Ad infection on endothelial permeability.
The effects of Ad infection per se on normal endothelial barrier
function were determined. The studies were made using Adgal for
infection, since expression of
-galactosidase in endothelial cells
is not expected to affect barrier properties. HMEC were grown to
confluency on microporous filters and infected with Ad
gal (5-100 MOI) or UV-Ad
gal (100 MOI) for 1, 2, or 3 days, and the transendothelial clearance rate of albumin was determined. The albumin
clearance rate of control noninfected HMEC was 0.021 ± 0.004 µl/min; infection of HMEC at 5, 50, and 100 MOI for 2 days resulted
in clearance rates not different from the control (Fig. 2A). As
a positive control, treatment with 100 µM
H2O2
increased the clearance rate to 0.056 ± 0.006 µl/min (Fig.
2A). Infection of HMEC at 100 MOI
for 1, 2, and 3 days also did not change the clearance rate relative to
control (Fig. 2B). HMEC infected
with UV-irradiated Ad
gal showed clearance rates not different from control (Fig. 2).
|
|
Ad-mediated overexpression of PKI.
Northern blot analysis indicated that infection of HMEC with AdPKI at
100 MOI for 24 h resulted in a single transcript hybridized with the
PKI-specific probe (Fig.
4, top, lane
2). However, mRNA was not detectable after 4 h of
infection (data not shown). The control noninfected HMEC
(lane 1) and HMEC infected with
AdNull (lane 3) showed absence of
PKI transcript. In Fig. 4,
bottom, equal loading of RNA of the
three groups is illustrated by the 28S rRNA band.
|
Effects of overexpression of PKI on endothelial permeability.
Initial studies were carried out to determine the effects of increased
intracellular cAMP on endothelial permeability in HMEC in response to
thrombin activation. A combination of forskolin (direct activator of
adenylyl cyclase) and IBMX (phosphodiesterase inhibitor) was used to
increase intracellular levels of cAMP in HMEC. Confluent
monolayers of HMEC grown on microporous filters were pretreated with F + I (Sigma Chemical) for 15 min, human -thrombin (0.5 µM)
was added, and albumin clearance rate was determined. The control
clearance rate was 0.015 ± 0.004 µl/min; thrombin treatment
increased the clearance rate to 0.040 ± 0.009 µl/min (Fig.
5). Pretreatment with F + I inhibited the
thrombin-induced increase in clearance rate (Fig. 5). The clearance
rates from F + I alone and the DMSO vehicle were not significantly
different from control.
|
|
Overexpression of PKI inhibits CRE-driven transcription of reporter
gene activity.
The function of PKI expressed in HMEC
was investigated using a reporter plasmid containing the firefly
luciferase gene under the transcriptional control of CRE
(pADneo2-C6-BGL) (24). HMEC were transiently transfected with
pADneo2-C6-BGL, and the activation of transcription was determined by
measuring luciferase activity. The transfected HMEC were treated with F + I for 2, 24, or 48 h, and cells were collected for luciferase
activity assay (see METHODS).
Results indicated that luciferase activity was greatest after 1 day of
F + I treatment and declined progressively by 48 h (Fig.
7A). The
control HMEC group (absence of F + I treatment) was consistently lower
in luciferase activity than the treated group.
|
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DISCUSSION |
---|
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Findings from this study indicate that overexpression of the PKI gene in endothelial cells abrogated the cAMP-mediated protective effects on the thrombin-induced increase in permeability and directly support the notion that the PKA signaling pathway was a predominant regulator functioning to prevent endothelial barrier dysfunction. Although the protective effects of increased intracellular cAMP are generally accepted to occur through activation of PKA, direct evidence supporting this idea has been limited. In one study, Stelzner et al. (50) reported that the pharmacological PKA inhibitor Rp-cAMPS reversed ~50% of the endothelial barrier enhancement produced by forskolin, an activator of adenylyl cyclase. They attributed this partial inhibition to nonspecific effects of Rp-cAMPS. However, an alternative explanation may be that forskolin (or increased cAMP) decreases endothelial permeability via PKA-dependent and -independent mechanisms. In the present study, overexpression of PKI in endothelial cells was effective in fully reversing the barrier-enhancing effects of F + I. PKI is an endogenous inhibitor of PKA and functions by competitive inhibition for the peptide substrate binding site on the catalytic subunit of PKA (52). The binding affinity of PKI is 0.2 nM and is highly selective and specific for PKA. The present findings provide strong support that cAMP-mediated barrier enhancement is regulated by predominantly PKA-dependent mechanisms.
The function of PKI was assessed by
its capacity to inhibit induction of luciferase activity in endothelial
cells transiently transfected with the reporter plasmid pADneo2-C6-BGL,
which contains six heterologous CRE sequences upstream of the
-globin minimal promoter (24). Cell lines transfected with this
plasmid showed luciferase induction that was specific to elevation of
cAMP levels (24). In this study, treatment of endothelial cells
transfected with the reporter plasmid with F + I induced a threefold
increase in luciferase activity over control. The induction of activity has been shown to involve a sequence of events initiated by increased cAMP, which lead to activation of PKA, phosphorylation of CRE-binding protein, binding of phosphorylated CRE-binding protein to CRE, and
activation of transcription. We found that overexpression of
PKI in endothelial cells abrogated
this transcriptional activation of luciferase activity, indicating that
the Ad-mediated PKI expression in
endothelial cells was functionally active in inhibition of PKA.
Two potential target substrates of PKA associated with regulating barrier function include Ca2+-calmodulin-dependent myosin light chain (MLC) kinase (MLCK) (15, 38, 43) and RhoA (12, 30). In endothelial cells, thrombin-induced barrier dysfunction is associated with cell rounding, intercellular gap formation, and increase in MLC phosphorylation (15, 39). Expression of the constitutively active catalytic domain of MLCK into Madin-Darby canine kidney epithelial monolayers resulted in a threefold increase in MLC phosphorylation, which was accompanied by increased paracellular permeability of solutes and decreased transepithelial resistance (23). Activated MLCK directly phosphorylates threonine-18 and serine-19 of MLC, which is related to isometric tension development and actin polymerization in endothelial cells (18). The specific target protein of RhoA, Rho kinase, is known to directly phosphorylate MLC as well as the myosin-associated MLC phosphatase, both processes contributing to the increased MLC phosphorylation (26, 28). In an in vitro study, inhibition of RhoA in endothelial cells by Clostridium botulinum C3 exoenzyme reduced the thrombin-induced barrier dysfunction and MLC phosphorylation (3), supporting the notion that the thrombin-induced barrier dysfunction occurred in part through activation of RhoA.
The recently cloned endothelial MLCK contains highly conserved phosphorylation sites for PKA, its phosphorylation resulting in reduced MLCK activity, which presumably provides the basis for protection against barrier dysfunction (16). PKA also terminates RhoA signaling by mediating the phosphorylation of the COOH-terminal domain of RhoA (30), providing an additional/alternative pathway by which MLC phosphorylation and endothelial permeability can be reduced. Several reports confirmed that elevation of intracellular cAMP in endothelial cells inhibited MLC phosphorylation in response to thrombin (15, 43) and histamine (38, 48). Recently, however, Moy and co-workers (37) observed that increased cAMP inhibited the thrombin-mediated decrease in transendothelial resistance, but it did not inhibit the increased MLC phosphorylation and tension development, suggesting that the promotion of barrier function was independent of MLC regulation. Thus the mechanisms by which PKA activation inhibits the mediator-induced increase in endothelial permeability are yet to be resolved.
Evidence also indicates that increased endothelial permeability may not be entirely accounted for by increased MLC phosphorylation (17, 39, 43, 47). Interestingly, Patterson et al. (43) showed that increased cAMP inhibited the endothelial barrier dysfunction in response to phorbol ester activation of protein kinase C, which occurred in the absence of increased MLC phosphorylation. These observations suggest that the mechanisms by which PKA confers protection against barrier dysfunction involve phosphorylation of other target substrates in addition to MLCK and RhoA. Substrates containing known PKA phosphorylation sites implicated in endothelial barrier function regulation include inositol 1,4,5-trisphosphate receptors (36, 57), filamin (21), and serine/threonine protein phosphatases (54). It will be important for future studies to identify the target substrates for phosphorylation by PKA and to determine their role in regulation of endothelial permeability.
Our findings indicate that the use of recombinant Ad vectors for gene
transfer in endothelial cells did not impair the barrier function, did
not result in cell toxicity, and showed a high gene transfer efficiency
and expression. The high efficiency of gene transfer is consistent with
reports from other investigators who have used similar recombinant Ad
vectors for in vitro infection (31, 51, 53) as well as for in vivo gene
transfer to a wide variety of target tissues (7). The finding that
infection of endothelial monolayers with Ad vectors did not impair
barrier function or induce cell cytotoxity indicated that Ad vectors
provide a particularly advantageous tool for studies of the effect of gene expression on endothelial barrier function. Furthermore, Piedimonte et al. (45) also documented a lack of direct effect of an
E1,
E3
, replication-deficient
recombinant Ad on tracheal barrier function. Thus the use of Ad vectors
for studies of barrier function is superior to other gene transfer
methods, such as the use of liposomes, electroporation,
CaPO4, and retroviral vectors,
which typically result in lower gene transfer efficiencies in addition
to cytotoxicity, which disrupts baseline barrier function.
In summary, infection of endothelial cells with the
E1,
E3
, replication-deficient
AdPKI resulted in overexpression of
PKI, which abrogated the cAMP-mediated
inhibition of the increased endothelial permeability in response to
thrombin. Overexpression of PKI also inhibited transcription of
the reporter gene luciferase, which is under the control of CRE,
indicating that the expressed PKI in
endothelial cells was functionally active. Furthermore, Ad-mediated
transgene expression of endothelial cells was highly efficient
(>95%), did not alter baseline endothelial barrier function, and was
not cytotoxic. Thus the results provide direct evidence that activation
of the PKA pathway is critically important in conferring protection
against mediator-induced endothelial barrier dysfunction.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Richard A. Maurer (Dept. of Cell Biology and Anatomy, School of Medicine, Oregon Health Sciences University, Portland, OR) for the generous gift of PKI-pUC13, Dr. Christopher B. Newgard (Dept. of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX) for providing pJM17 and pACCMV.pLpA, and Dr. Adolf Himmler (Ernest Boehringer Institut, Vienna, Austria) for pADneo2-C6-BGL.
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FOOTNOTES |
---|
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62649 (H. Lum) and HL-53623 (R. D. Green) and the American Lung Association of Metropolitan Chicago (H. A. Jaffe).
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.
Address for reprint requests and other correspondence: H. Lum, Dept. of Pharmacology, Rush Presbyterian St. Luke's Medical Center, 2242 W. Harrison St., Suite 260, Chicago, IL 60612 (E-mail: hlum{at}rush.edu).
Received 20 April 1999; accepted in final form 11 May 1999.
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