1 Department of Medicine, University of Florida, and 3 Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608; and 2 Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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We examined
whether nitric oxide (NO)-induced inhibition of thioredoxin (Thx)
expression is regulated by a mechanism mediated by a transcription
factor, i.e., nuclear factor-B (NF-
B), in cultured porcine
pulmonary artery endothelial cells (PAEC) and in mouse lungs. Western
blot analysis revealed that I
B-
content was reduced by 20 and
60% in PAEC exposed to 8.5 ppm NO for 2 and 24 h, respectively. NO
exposure also caused significant reductions of cytosol fraction p65 and
p52 content in PAEC. The nuclear fraction p65 and p52 contents were
significantly reduced only in PAEC exposed to NO for 24 h. Exposure to
NO resulted in a 50% reduction of p52 mRNA but not of the I
B-
subunit. DNA binding activity of the oligonucleotide encoding the
NF-
B sequence in the Thx
gene was significantly reduced in PAEC exposed to NO for
24 h. Exposure of mice to 10 ppm NO for 24 h resulted in a significant
reduction of lung Thx and I
B-
mRNA and protein expression and in
the oligonucleotide encoding Thx and NF-
B/DNA binding. These results
1) demonstrate that the effects of
NO exposure on Thx expression in PAEC are comparable to those observed
in intact lung and 2) suggest that reduced expression of the NF-
B subunit, leading to reduced
NF-
B/DNA binding, is associated with the loss of Thx expression in
PAEC and in intact mouse lungs.
nuclear factor-B; transcription factor; gene regulation; mouse
lung; lung endothelium
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INTRODUCTION |
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NITRIC OXIDE (NO), a free radical gas produced by vascular endothelial and other mammalian cells, plays a critical role in lung physiology and pathophysiology (5, 14). Despite the important role of NO in the regulation of vascular tone in the pulmonary circulation, in vivo and in vitro studies indicate that NO is cytotoxic to a number of cells, including vascular endothelial cells (24, 27, 29, 31). For example, NO has been reported to exert a toxic role during such diverse pathological events as chemically induced pulmonary edema, ischemia-reperfusion injury, allograft transplant rejection, and inflammatory lung disease in which endothelial cell dysfunction is common (6, 18, 39). We recently reported that 1) exposure of porcine pulmonary artery endothelial cells (PAEC) to NO significantly reduced the catalytic activity of the constitutive (endothelial cell) isoform of NO synthase (ecNOS) (31), 2) disulfide reducing chemicals, e.g., dithiothreitol or 2-mercaptoethanol or enzymatic reduction of disulfide by thioredoxin/thioredoxin reductase (Thx/Thx-R), but not by glutaredoxin, restored NO-induced loss of ecNOS catalytic activity (30, 31), and 3) exposure to NO significantly reduced the expression of the redox regulatory proteins Thx and Thx-R in PAEC (42). Although the effect of NO on lung Thx expression is not known, the level of lung Thx content is critical for maintaining redox regulation of proteins under oxidative stress.
Thx plays a critical role in the regulation of catalytic activity and
the function of redox-sensitive proteins, enzymes, receptors, and
transcription factors, including nuclear factor-B (NF-
B) (1, 17,
35). Rel family transcription factors
NF-
B1 and NF-
B2 are involved in the
expression of a wide variety of genes, including
ecNOS and
Thx genes (20, 25, 26). NF-
B exists primarily as a heterodimer composed of 65- and 50-kDa (p65 and p50,
respectively, NF-
B1) or
52-kDa (p52, NF-
B2) subunits
that is sequestered in the cytoplasm with the inhibitory subunit
I
B-
, I
B-
, I
B-
, or Bcl-3 (11, 38, 40). Homodimers of
p50 and p65 have also been reported (4). Activation of the NF-
B
complex involves dissociation of the heterodimer complex from the I
B subunit in the cytosol and translocation to the nucleus, which results
in DNA binding and activation of gene expression (40). NO has been
shown to inhibit transcription factor/DNA binding in mammalian cells
(23, 28, 33). In the present study we report
1) the in vitro effects of NO
exposure on NF-
B subunit dissociation, translocation, and expression
and on NF-
B/DNA binding activity of the Thx promoter in cultured
PAEC and 2) the in vivo effects of
NO exposure on Thx and I
B-
expression and NF-
B/DNA binding to
the Thx promoter in mouse lungs.
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MATERIALS AND METHODS |
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Tissue culture. Endothelial cells were isolated from the main pulmonary artery of 6- to 7-mo-old pigs and propagated in monolayer cultures, as reported by Patel and Block (30). Fifth- to seventh-passage PAEC in postconfluent monolayers maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) with 4% fetal bovine serum (HyClone Laboratories, Logan, UT) and antibiotics were used for all experiments.
Exposure of PAEC and mice to NO. Confluent PAEC monolayers were exposed to a continuous flow of 8.5 ppm NO gas premixed with 5% CO2 and air for 2 and 24 h, as previously described (32, 42). Exposure to NO for 24 h under these conditions did not alter the pH (7.4) of the culture medium. NO2 was not detectable in the premixed gas or in the exposure chamber.
For the mouse exposure study, male C57BL/6J mice (26-28 g; Jackson Laboratories) were continuously exposed to 10 ppm NO for 24 h. Animal procedures met standards of the University of Texas Medical Branch (Galveston) Animal Care and Use Committee. Mice had free access to food and water throughout the exposure period. With use of mass flow controllers, a high concentration of NO (1,000 ppm NO in nitrogen; Liquid Carbonic, Pasadena, TX) was bled into a stream of compressed air to achieve the desired concentration and an exposure chamber turnover rate of 15 volume changes per hour. Chamber concentrations of NO and NO2 were continuously monitored using a environmental chemiluminescence NOx analyzer (model 42, Thermo Environmental, Franklin, MA) and were maintained at 10 ppm NO. During exposure, NO2 concentrations averaged 0.17 ± 0.04 ppm. Immediately on removal from the chamber, animals were anesthetized with pentobarbital sodium (60 mg/kg ip), with the depth of anesthesia verified via foot pinch. A midline thoracotomy was performed to expose the lungs, and after transection of the abdominal aorta, the lungs were removed en bloc and immediately frozen in liquid nitrogen for later analysis. NO-exposed PAEC or lungs were used for the isolation of cytosol and nuclear fractions, Western blot analysis, RNA extraction, and protein determination. Lungs were also used for isolation of total membrane fractions and for assays of NO synthase (NOS) activity.Isolation of cytosol, total membrane, and nuclear fractions.
Lung cytosol and total membrane fractions were isolated to compare the
in vivo effect of NO on NOS activity with the previously reported in
vitro effect of NO on ecNOS activity in PAEC (32). PAEC cytosol and
nuclear fractions and whole lung nuclear fractions were isolated to
examine expression of NF-B subunits and NF-
B/DNA binding
activity. For isolation of cytosol fractions from PAEC and cytosol and
total membrane fractions from intact lung, control and NO-exposed cell
monolayers and lungs were washed twice and homogenized using a
motor-driven Potter-Elvehjem Teflon-glass homogenizer with 10 strokes
at 4°C in buffer A (50 mM
Tris · HCl, pH 7.4, containing EDTA and EGTA at 0.1 mM each, 1 mM phenylmethylsulfonyl fluoride, and leupeptin at 1.0 mg/l). The homogenates were centrifuged at 100,000 g for 60 min at 5°C in an
ultracentrifuge (model L3-50, Beckman, Irvine, CA), the total membrane
pellets were resuspended in buffer A,
and the supernatants (cytosol) were collected separately as described
previously (43).
Measurement of lung NOS activity. Exposure to NO is known to reduce ecNOS activity in PAEC (30). We examined whether exposure to NO has similar effects on calcium-dependent NOS activity in the total membrane fraction of mouse lungs. Total membrane and cytosol fraction NOS activity or cytosol NOS activity alone was measured by monitoring the formation of L-[3H]citrulline from L-[3H]arginine (8). For total NOS activity, membranes (100-120 µg of protein) were incubated (total volume 0.4 ml) in buffer A containing 1 mM NADPH, 100 nM calmodulin, 10 µM tetrahydrobiopterin, 2.5 mM CaCl2, and 5 µM combined L-arginine and purified L-[3H]arginine (0.6 µCi, specific activity 69 Ci/mmol; NEN, Boston, MA) for 30 min at 37°C. Calcium-independent NOS, i.e., inducible NOS (iNOS), activity was measured under identical conditions in a similar incubation mixture containing 1 mM EDTA and no CaCl2. Blanks were incubated under identical conditions but in the absence of total membrane protein. Purification of commercially available L-[3H]arginine was carried out as described previously (31). Total NOS activity was determined after subtraction of the activity of the blank. Calcium-dependent NOS activity was determined after subtraction of iNOS activity from total NOS activity. The specific activity of NOS is expressed as picomoles of L-citrulline per minute per milligram of protein.
Western blot analysis of IB-
, p65,
and p52 proteins.
The effects of NO exposure on expression of PAEC cytosol and nuclear
fraction I
B-
, p65, and p52, as well as on lung expression of Thx,
were examined. PAEC cytosol and nuclear fractions as well as mouse lung
homogenate proteins (40 µg each) from control and NO-exposed PAEC and
lungs were separated by 12% SDS-PAGE and electroblotted onto a
nitrocellulose membrane (Bio-Rad, Richmond, CA), as described previously (42). The blots were incubated in blocking solution [0.2% nonfat milk in TBS (20 mM Tris · HCl, pH
7.5, and 500 mM NaCl)] and then hybridized with anti-I
B-
,
anti-p65, or anti-p52 (Santa Cruz Biotechnology, Santa Cruz, CA)
monoclonal antibodies (1:2,000 dilution) or human anti-Thx monoclonal
antibody (American Diagnostica, Greenwich, CT) (1:1,000 dilution) in
antibody buffer (0.2% nonfat milk and 0.1% Tween 20 in TBS) at room
temperature for 1 h. After they were washed with buffer (0.1% Tween 20 in TBS), the membranes were incubated in 1:3,000-diluted anti-rabbit IgG, alkaline phosphatase-linked whole antibody (Immunstar
chemiluminescent protein detection system, Bio-Rad) for 1 h. The
immunoreactive bands were visualized by enhanced chemiluminescence
(Amersham, Arlington Heights, IL) with Kodak XAR-5 film for 5-15
min. The blots were scanned using the Fluor-S MultImager system
(Bio-Rad) to quantify these protein contents.
RNA isolation and RNase protection assay.
Total RNA was extracted directly from control and NO-exposed PAEC and
mouse lung by use of the RNAgents Total RNA Isolation System (Promega,
Madison, WI). RNA contents of the extracts were determined by measuring
levels of 18S rRNA as an internal standard, since the gene expression
of 18S RNA was not altered by NO exposure (unpublished data). mRNA was
extracted from the total RNA preparations by use of the PolyATtract
mRNA isolation system (Promega) according to the manufacturer's
instructions. Digoxigenin-UTP (DIG)-labeled RNA probes for IB-
(600 bp), Thx (330 bp), and p52 (120 bp) were transcribed in vitro from
Pvu II-digested pBS-I
B (kindly provided by Dr. Stephen Haskill, University of North Carolina, Chapel
Hill, NC), linearized pThx-A, as described by Zhang et al. (42), and
linearized pTRI-p52 (obtained from reagent donor Dr. Gary Nabel through
the AIDS Research and Reference Reagent Program, Division of Acquired
Immune Deficiency Syndrome, National Institute of Allergy and
Infectious Diseases), respectively. The RNase protection assay was
carried out to determine the mRNA levels with use of an RNase
protection kit (Boehringer Mannheim Biochemicals, Indianapolis, IN)
(19, 44). mRNA extracts (5 µl, 200 ng/µl) from control and
NO-exposed PAEC or whole mouse lungs and 1.5 µl of DIG-labeled I
B,
Thx, or p52 RNA probe (40 ng/µl each) were mixed, denatured, and
incubated at 45°C overnight. After RNase treatment, the partial
RNA:RNA hybrids were subjected to electrophoresis in a polyacrylamide
(4%)-7 M urea gel at ~30 W for 2.5 h in Tris-borate buffer. After
being blotted onto a nylon membrane (Zeta-Probe GT, Bio-Rad), the
digestion products were immunodetected with anti-digoxigenin-AP and the
chemiluminescence substrate CSPD (Genius Labeling and Detection kits,
Boehringer Mannheim), as described by the manufacturer, and then
exposed to Kodak BioMax film for 5-15 min. Quantitation of mRNA
for the I
B, Thx, and p52 was performed using the Fluor-S MultiImager
system (Bio-Rad). The levels of specific mRNA content were standardized
to the
-actin mRNA (Ambion, Austin, TX) levels, which are not
affected by NO exposure.
Electrophoretic mobility shift assay.
The DNA band-shift analysis of PAEC and mouse lung nuclear fractions
was performed as reported by Baldassarre et al. (3). Briefly, the Thx
NF-B double-stranded oligonucleotide (5'-AGA CCT GGG ACT CTC
CCT CCC AGC-3') was synthesized by the Interdisciplinary Center
for Biotechnology Research, University of Florida (Gainesville, FL).
The nuclear fraction (10 µg protein) was incubated with 1 µg of
poly(dI-dC) and 0.1 µg of
poly-L-lysine in 20 mM HEPES
buffer, pH 7.6, containing 1 mM EDTA, 10 mM
(NH4)2SO4, 0.2% (wt/vol)
Tween 20, and 30 mM KCl for 15 min at room temperature. At the end of incubation, 5× loading buffer was added, and the samples were electrophoresed in a native 7% polyacrylamide gel in buffer (pH 8.5)
containing 90 mM Tris, 90 mM boric acid, and 2 mM EDTA. The DIG
nucleotide/NF-
B and free probes were electroblotted onto a nylon
membrane and immunodetected with anti-digoxigenin-AP and the
chemiluminescence substrate CSPD (DIG gel shift kit, Boehringer Mannheim), as described by the manufacturer, and then exposed to Kodak
XAR-5 film for 5-15 min. Specificity of Thx
oligonucleotide/NF-
B binding was determined by using a 10-fold
excess of unlabeled oligonucleotide as well as by supershift assay with
p65 antibody. Densitometric analysis for the NF-
B/DNA band was
performed using the Fluor-S MultiImager system.
Statistical analysis.
Statistical significance for the effects of NO exposure on the
IB-
, p65, and p52 protein contents, I
B-
, p52, and Thx mRNA levels, Thx oligonucleotide-NF-
B/DNA binding activity, and NOS activity was determined using Student's paired
t-test (41). Values are means ± SE
for n experiments.
P < 0.05 was taken as significant.
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RESULTS |
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Exposure of PAEC monolayers to 8.5 ppm NO for 2 or 24 h had no significant effect on endothelial cell morphology assessed by phase-contrast microscopy. Similarly, protein contents of cytosol and membrane fractions from PAEC exposed to NO for 2 and 24 h were comparable to controls (data not shown). In addition, exposure of mice to 10.1 ± 0.2 (SD) ppm NO for 24 h produced no alterations in lung weight or overt signs of gross pathology (e.g., pulmonary edema).
Effect of NO exposure on mouse lung NOS activity and Thx mRNA and
protein expression.
Because NO exposure results in reduction of ecNOS activity and Thx
expression in cultured PAEC (32, 42), we examined the in vivo response
to NO exposure by measuring total membrane fraction calcium-dependent
NOS activity and Thx mRNA and protein expression in the lungs of mice
exposed to NO gas. Total membrane fraction calcium-dependent NOS
activities in control and NO-exposed lungs were 3.38 ± 1.0 and 1.21 ± 0.6 nmol
L-citrulline · min1 · mg
protein
1
(P < 0.001, n = 4), respectively.
Calcium-dependent NOS activity in cytosolic fractions from control and
NO-exposed lungs was not detectable under our experimental conditions.
Exposure of mice to 10 ppm NO gas for 24 h resulted in decreased
expression of lung Thx mRNA (Fig. 1,
A and
B) and protein (Fig.
1C) contents (P < 0.05 for both).
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Effect of NO exposure on NF-B subunits
I
B-
, p65, and p52 in PAEC.
To examine whether NO exposure alters NF-
B subunit dissociation and
translocation, cytosol and nuclear fraction I
B-
, p65, and p52
protein levels were measured. As shown in Fig.
2, exposures to NO for 2 and 24 h reduced
the I
B-
protein levels in the cytosol by 20%
(P < 0.05) and 60%
(P < 0.01), respectively, compared
with controls. As shown in Fig. 3, exposure
of PAEC to NO for 2 and 24 h significantly decreased the protein levels
of p65 in the cytosol fractions (P < 0.01 for both). In addition, p65 protein content in the nuclear
fraction of PAEC exposed to NO for 24 h, but not for 2 h, was
significantly (P < 0.01) reduced
compared with controls. The p52 protein contents in the cytosol and
nuclear fractions of cells exposed to NO for 24 h, but not for 2 h,
were significantly reduced (P < 0.01 for both; Fig. 4). These results suggest
that exposure to NO reduces the translocation and/or expression of the
NF-
B subunits in PAEC.
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Effect of NO on IB-
and p52 mRNA
expression.
To determine whether the NO-induced reductions of NF-
B subunit
proteins in the cytosol and/or the nuclear fraction are associated with
reduced mRNA expression for these proteins, we monitored I
B-
and
p52 mRNA contents in PAEC exposed to NO for 24 h, in control PAEC, and
in the lungs of mice. As shown in Fig. 5,
I
B-
mRNA content in NO-exposed cells was slightly, but not
significantly, reduced, whereas the lung I
B-
mRNA contents of
mice exposed to NO were significantly
(P < 0.01) reduced compared with
controls. The p52 mRNA content in NO-exposed cells was significantly
(P < 0.01) reduced compared with
controls, whereas the p52 mRNA content of mice exposed to NO was
comparable to controls (Fig. 6).
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Effect of NO exposure on Thx-oligonucleotide/NF-B
binding activity.
Alterations in the levels of the NF-
B subunits p65/p52 and/or the
extent of their nuclear translocation have been shown to influence
NF-
B/DNA binding. Consequently, we examined the effects of
transcription factor NF-
B/DNA binding on Thx gene
expression. The oligonucleotide-NF-
B/DNA binding activity was
standardized by gel mobility shift assay with use of a 10-fold excess
of unlabeled oligonucleotide, as well as by supershift assay with use
of p65 antibody (Fig.
7A). As
shown in Fig. 7, B and
C, exposure to NO gas for 24 h
significantly reduced Thx-oligonucleotide-NF-
B/DNA binding activity
in PAEC and in the lungs of mice compared with controls
(P < 0.01 for both). However,
Thx-oligonucleotide-NF
B/DNA binding activity was comparable to
controls in PAEC exposed to NO gas for 2 h (data not shown). This
NO-induced reduction of NF-
B/DNA binding activity may account for
the NO-induced reductions of Thx mRNA and protein expression in PAEC
and mouse lungs.
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DISCUSSION |
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We recently reported that exposure to NO resulted in the loss of
catalytic activity of NOS as well as reduced Thx and Thx-R mRNA and
protein expression in PAEC (32, 42). The results of the present study
demonstrate for the first time that exposure to NO also results in loss
of NOS catalytic activity and Thx mRNA and protein expression in the
lungs of mice exposed to NO gas and suggest that NO-induced reduction
of Thx gene expression is mediated
through NF-B modulation in PAEC and mouse lung. NO-induced reduction
in Thx oligonucleotide-NF-
B binding activity was associated with
decreased expression and/or impaired translocation of NF-
B subunits
from cytosol to nuclear fractions in PAEC and intact lungs. This
indicates that NO-induced diminished expression of Thx in PAEC and
intact lung is regulated through an NF-
B-mediated mechanism.
Alternatively, this mechanism may also be Thx dependent with respect to
the level of Thx expression and/or its redox status.
NF-B-activated gene expression requires dissociation of the
inhibitory protein I
B-
in the cytosol followed by translocation of the NF-
B dimer (p65/p52) to the nucleus. A number of
extracellular stimuli, including oxidative stress, viral and bacterial
infection, drugs, and environmental chemical agents, physical stress,
and biological mediators such as inflammatory cytokines have been implicated in alterations of NF-
B/DNA binding activity and gene expression (11, 33, 38, 40). Although a number of studies have reported
that exposure to NO or NO donors modulates NF-
B/DNA binding activity
in various mammalian cells (12, 23, 28, 33, 37), the results of the
present study establish a specific association between NO-induced
NF-
B modulation and reduced binding to the DNA sequences for
Thx gene transcription in PAEC and
mouse lung. These observations are consistent with and support our
previous report in which cultured PAEC were used (42), and the present results in mouse lungs demonstrate that NO-induced diminished expression of Thx mRNA and protein occurs in vivo and is mediated by a
transcriptional mechanism.
The precise mechanisms responsible for the NO-induced reduced
expression of IB-
, p65, and p52 subunit proteins and of I
B-
and p52 mRNA contents as well as for the NO-induced reduction in the
nuclear translocation of the p65/p52 dimer in the present study are not
known. However, it is possible that NO-induced inhibition of total
protein synthesis or synthesis of specific proteins such as I
B-
,
p65, and p52 or the 100-kDa precursor protein (p100) of p52 may account
for the reduced expression of these subunits in PAEC and intact lung,
inasmuch as NO and NO-generating agents are known to inhibit total as
well as specific protein synthesis in a variety of mammalian cells (13,
21, 22). Precise concentrations of NO generated from donors
(100-400 µM) were not characterized in these studies.
NO is also known to interact with protein sulfhydryls to form
S-nitrosothiols, resulting in inactivation of a number of
redox-sensitive proteins, enzymes, receptors, and transcription
factors, including NF-B (1, 2, 17, 29, 32). Thus direct oxidation of NF-
B subunit proteins by NO may result in reduced dissociation, translocation, and/or DNA binding activity. Several other mechanisms critical for dissociation of the inhibitory protein I
B-
from NF-
B may also be partially responsible for reduced translocation of
the p65/p52 dimer to the nucleus. For example, a number of factors,
including phosphorylation, proteolytic degradation, and activation of
I
B-specific kinase, are known to play a role in NF-
B activation
(10, 15, 23), and NO may affect any one of these.
The implications of our observations are significant for NO-induced
modulation of the redox-sensitive transcription factor NF-B and its
component proteins, which play a critical role in the regulation of a
variety of genes. Similarly, tissue level of Thx may be critical in
maintaining redox status of a variety of enzymes in the signal
transduction cascade. The tissue level of Thx is especially important,
since Thx plays a critical role in the cellular defense against
oxidative stress, including protection against NO-induced loss of ecNOS
activity in PAEC (32). This is particularly important in pathological
conditions where NO plays a role, including septic shock, organ
transplant rejection, inflammation, radiation damage, chemical- or
drug-induced injury, and atherosclerosis. In addition, exogenous
administration of inhaled NO gas for extended periods is commonly used
to restore lung hemodynamics and gas exchange in newborn or adult
patients with primary pulmonary hypertension, respectively (34, 36), and adult respiratory distress syndrome (7, 16). Therapeutic use of NO
gas has the potential to alter the function of a number of
redox-sensitive proteins in lung cells. Thus maintaining Thx levels is
critical for regulation of lung cell redox regulatory processes and
pulmonary function.
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ACKNOWLEDGEMENTS |
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We thank Bert Herrera for tissue culture assistance, Di-hua He for technical assistance, Addie Heimer for manuscript preparation, and Janet Wootten for excellent editorial help.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-58679 (to J. M. Patel) and HL-54679 (to E. M. Postlethwait) and by the Department of Veterans Affairs Medical Research Service.
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: J. M. Patel, Research Service (151), Dept. of Veterans Affairs Medical Center, 1601 SW Archer Rd., Gainesville, FL 32608-1197 (E-mail: Pateljm{at}medicine.ufl.edu).
Received 18 December 1998; accepted in final form 28 May 1999.
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