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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Hematology/Oncology, Department of
Medicine, Center for Cardiovascular and Molecular Medicine, Department
of Psychiatry, State University of New York Downstate Medical Center,
Brooklyn, NY.
Oxygen deprivation (hypoxia) is a consistent component of ischemia
that induces an inflammatory and prothrombotic response in the
endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O2) increases
messenger RNA and protein levels of transforming growth factor- One of the earliest responses of endothelium to
hypoxia is inflammation, and the balance between positive and negative
regulators of this inflammatory response is a determinant of
endothelial adaptation to hypoxia. Proinflammatory endothelial
responses induced by hypoxia involve production of cytokines,
chemokines,1-3 tissue factor,4 and
plasminogen activator inhibitor-1 (PAI-1).5 Recent studies
indicate that activation of the transcription factor early growth
response-1 (Egr-1) by hypoxia is responsible for the procoagulant
profile and vascular remodeling that ultimately result in the tissue
damage associated with ischemia.4 Adaptive responses to
hypoxia are also regulated at a transcriptional level, which is best
exemplified by activation of the transacting protein hypoxia-inducible
factor-1 (HIF-1), which stimulates differential expression of specific
genes and results in increases in glucose uptake, glycolytic enzyme
production, and angiogenesis.6,7
The transforming growth factor- Three mammalian isoforms of TGF- Recent studies of human endothelial cells and in vivo animal studies
have shown that Smad proteins regulate endothelial responses to
mechanical stress such as shear31,32 and to inflammatory stress such as exposure to bacterial lipopolysaccaride.33
It seemed reasonable, therefore, to test the hypothesis that TGF- Cell cultures
RNase protection assay
TGF- -responsive mink lung epithelial cells (MLECs) stably
transfected with the expression construct p800neoLUC containing a
truncated PAI-1 promoter fused to the firefly luciferase reporter gene
were kindly supplied by Dr Daniel B. Rifkin (New York University School
of Medicine, New York, NY). Cells were maintained in Dulbecco modified
Eagle medium containing 10% fetal calf serum, 2 mM L-glutamine, and
antibiotics (100 U/mL penicillin G, 100 µg/mL streptomycin, 250 µg/mL geneticin; Gibco). To assay bioactive and total TGF- 2 levels
in HUVEC supernatants, MLECs were detached with trypsin, washed, plated
at 1.6 × 105 cells per well in a 2 mL volume in 24-well
tissue culture plates (Costar), and were allowed to attach for 18 hours
at 37°C in 5% CO2. Medium in the wells was replaced by 2 mL of the following, placed in triplicate wells: control medium;
control medium containing increasing concentrations of recombinant
TGF- 2 to generate a standard curve; 1:10 dilution of conditioned
HUVEC medium to measure bioactive TGF- ; and 1:10 dilution of
conditioned HUVEC medium activated for 10 minutes by heating at 80°C
to measure total TGF- . Incubations were continued for 18 hours at
37°C, and MLEC lysates from each well were prepared using reporter
lysis buffer (Luciferase Assay System, Promega, Madison, WI).
Luciferase activity was measured as relative light units (RLUs; model
TD-20/20 luminometer, Promega), which were converted to TGF-
activity (picomoles) using the TGF- 2 standard curve.20
Total protein content of supernatants was quantitated using Bio-Rad
protein assay reagent (Bio-Rad, Hercules, CA),36 and
TGF- 2 levels obtained by the luciferase assay from each well were
normalized to protein. Mean RLUs from triplicate wells (which were
within 5%-7% of one another) were converted to picomoles of TGF- 2.
This assay is sensitive to all 3 isoforms of TGF- ; therefore, to
specifically quantitate total TGF- 2 in hypoxic and nonhypoxic
supernatants, aliquots of the identical supernatants used in MLEC assay
were acid-activated and subjected to enzyme-linked immunosorbent assay
(ELISA) (Quantikine, R&D Systems) as previously
described.37 Results (not shown) confirmed that TGF- 2
levels were increased by 2-fold in hypoxic supernatants. Because
measurement of bioactive TGF- s by ELISA is not yet reliably accomplished by existing commercial kits, this assay can only confirm
the increase in total TGF- 2 determined by the MLEC assay.
Nuclear run-on assay for TGF- 2 in HUVECs was determined
as described by Greenberg and Ziff38 in nuclei prepared
from HUVECs cultured in either normoxic or hypoxic culture conditions. Briefly, a suspension of 6 × 109 nuclei in 200 µL
glycerol (40%) in 50 mM Tris-HCl buffer (pH 8.3, with 5 mM
MgCl2 and 0.1 mM EDTA) was mixed with 2 × reaction buffer
(10 mM Tris-HCl [pH 8]; 5 mM MgCl2; 0.3 M KCl; 5 mM
dithiothreitol; 1 mM each ATP, CTP, GTP; and 100 µCi [3.7 × 106 Bq] [32P]UTP [1.1 × 1014 Bq, 3000 Ci/mM; PerkinElmer Life
Sciences, Boston, MA]). After incubation for 30 minutes at
30°C, [32P]-labeled RNA was extracted twice with
phenol/chloroform/isoamyl alcohol (25:24:1) and 50 µg transfer RNA,
and 10% trichloracetic acid (TCA) were added. The mixture was filtered
through a 0.45-µm Millipore filter (Millipore, Bedford, MA).
Nuclear RNA from the filter was released by treatment with 20 mM HEPES
buffer (pH 7.5, with 5 mM MgCl2 and 1 mM CaCl2)
and deoxyribonuclease for 30 minutes at 37°C, addition of 45 µL of
0.5 M EDTA and 68 µL of 20% SDS, and incubation at 65°C for 10 minutes. Nuclear RNA was digested with proteinase K, extracted with
phenol/chloroform/isoamyl alcohol, and ethanol-precipitated at 20°C
overnight. TGF- 2, Glut-1, and GAPDH complementary DNA probes have
been described.39-41 RNA was hybridized for 36 hours at
65°C to linearized plasmid DNA (TGF- 2, Glut-1, GAPDH) immobilized
on nitrocellulose membranes.41 Filters were washed in SSC
(2 ×) at 65°C 3 times for 30 minutes each, incubated at 37°C with
80 µg ribonuclease A, washed again, air dried, and exposed to DuPont
reflection film (DuPont Pharmaceuticals, Wilmington, DE), with an
intensifying screen, at 80°C, and also by exposure to
PhosphorImager. For quantification of TGF- 2 transcripts and
determination of the fold increase in transcript levels in hypoxic
HUVECs compared with control, the TGF- 2/GAPDH signal ratio from
hypoxic HUVECs was divided by the TGF- 2/GAPDH signal obtained from
normoxic HUVECs.
Plasmid constructs Plasmid constructs containing deletion mutants of the human TGF- 2 gene promoter linked to a chloramphenicol acetyl transferase (CAT) gene were generously donated by Dr S.-J. Kim.42
TGF- 2/CAT constructs pB2-778, pB2-257, and pB2-40 were generated by
polymerase chain reaction using genomic DNA containing the 5'-flanking
DNA and the first exon of the TGF- 2 gene as template with different 5'-specific oligonucleotide primers, spanning from 778 to 40, and a
common 3'-specific oligonucleotide primer at +63 relative to the
transcription initiation site; amplified DNAs were cloned into the
promoterless pGEM4/SV0CAT vector as described.42 The p3TP-Lux construct (a generous gift from Dr Joan Massagué,
Memorial Sloan-Kettering Cancer Center, New York, NY) is a well
described and widely used artificial promoter construct that was
designed to have maximal responsiveness to TGF- .23 This
plasmid has 3 AP-1 sites corresponding to the collagenase promoter,
concatemerized 5' to a 400-nucleotide region of the PAI-1 promoter,
followed by 70 base pairs (bp) of the adenovirus E4
promoter.43 Activity of this construct is increased
significantly by TGF- as well as by combined overexpression of Smad3
and Smad4. Smad4 is required for this transcriptional activity because
3TP-Lux is inactive in Smad4-null cells and is rescued by Smad4
expression.23 Expression vectors for Smad3 and Smad4 that
contain the FLAG epitope tag at the amino domain have been described
previously25 and were generously supplied by Drs Rik
Derynck and Ying Zhang (University of California, San Francisco, CA).
Transfection of recombinant plasmids and reporter gene assays For transient transfections, plasmids containing 5' TGF- 2
promoter sequences and p3TP-Lux containing the luciferase reporter gene, with indicated expression vectors or corresponding empty constructs, were introduced by electroporation into early passage (second or third) HUVECs that were 70% confluent, as previously described.35 Briefly, 6 × 106 HUVECs, HepG2
(HB-8065, American Type Culture Collection [ATCC], Manassas,
VA), A549 (CCL-185, ATCC), and MDA-MB-468 (HTB-132, ATCC) were
resuspended in 600 µL respective growth media and reacted with 30 µg reporter plasmid DNA along with the indicated Smad expression
plasmids or corresponding empty constructs (14 µg) and 0.5 µg
plasmid cytomegalovirus -galactosidase (pCMV -gal; Promega)
(included in each transfection as internal control) for 10 minutes at
4°C. When multiple plasmids were cotransfected (eg, reporter and
expression plasmids), the total amount of DNA was kept constant by
supplementing the samples with empty expression vectors.
Electroporation was performed using 220 V and 960 microfarads in a
Bio-Rad Gene Pulser Electroporation System. Transfected cells were
divided into 2 equal aliquots and cultured in 60-mm plates in
endothelial cell growth medium for 6 hours, after which one of each
pair of plates was placed in hypoxic conditions while the duplicate
plate remained in nonhypoxic culture conditions for indicated periods.
Adherent cells were washed with phosphate-buffered saline 36 hours
after transfection, dislodged from plates using a rubber policeman, and
the CAT assay performed at assay conditions predetermined to be within
the linear range for CAT activities of the samples (data not shown).
Luciferase activity was determined using the Luciferase Assay System
(Promega). To ensure that the same amount of protein was analyzed, the
protein concentration of each sample was determined according to the
Bradford assay,36 and transfection efficiency was
determined by -gal activity. CAT/ -gal/protein or
RLU/ -gal/protein ratios for hypoxic and nonhypoxic transfectants
were compared to determine the fold induction by hypoxia. The displayed
error bars in Figures 5 and 6, which are each representative of at
least 3 independent experiments, represent ± 1 SD of the mean.
Electrophoretic gel mobility shift assays Fresh medium with or without TGF- 2 (12.5 ng/mL) was added to
70% confluent HUVECs, and half of the duplicate flasks were placed in
hypoxic conditions. At the end of the indicated incubation periods,
nuclear extracts were prepared from hypoxic and nonhypoxic HUVECs as
described by Andrews and Faller.44 Protein concentrations were determined by the Bio-Rad assay with bovine serum albumin standards; final protein concentration of nuclear extracts was usually
between 5 and 10 mg/mL. Aliquots were stored at 70°C until use. A
double-stranded oligonucleotide corresponding to the 26-bp
sequence29 5'-AGTATGTCT-AGACTGA-3'
harboring the underlined consensus palindromic SBE was used as probe.
The SBE probe was end-labeled with [ -32P]dATP using
the Klenow fragment of DNA polymerase I for gel shift studies, and 50 to 100 pg (1 × 104-2 × 104 cpm) was
incubated with 7 µg nuclear extract and 2 to 8 µg double-stranded poly(dIdC) in 20 µL of 10 mM HEPES (pH 7.9), 50 mM KCl, 5 mM
MgCl2,1 mM EDTA, 1 mM dithiothreitol, and 5% wt/vol
glycerol for 30 minutes at 4°C after preincubation of all components
except probe for 15 minutes. For competition experiments, 20 to 500 molar excess of the indicated unlabeled oligonucleotide was added to
the binding reaction mixture 5 minutes prior to probe addition. The
following cold oligonucleotides were used for competition for nuclear
extract binding to the probe: an 18 bp sequence corresponding to
nucleotides 1 to 18 of the erythropoietin gene hypoxia-responsive
enhancer45 5'-GCCCTACGTGCTGTCTCA-3' harboring
the HIF-1-binding core sequence (in italics); a 39 bp sequence
corresponding to 77 to 40 bp of the TGF- 2 promoter with respect
to transcriptional start site 5'-CCTAGCACGTCACTTTGTTGAAGGCAGACACGTGGTTCA-3'
harboring an underlined CAGA box and an HIF-1-binding core
sequence46; and SBE. For supershift assays, each antibody
(rabbit antihuman Smad2, anti-Smad3 [Zymed, South San Francisco, CA],
rabbit anti-Smad4 [Upstate Biotechnology, Lake Placid, NY], or rabbit
immunoglobulin G) was added to the binding reactions 15 minutes prior
to the probes and incubated for another 30 minutes. After
preelectrophoresis for 1 hour, 4% polyacrylamide gel electrophoresis
was performed in 0.5 × Tris-borate buffer/1% glycerol without EDTA
at 180 V for 2 hours at 4°C. The gel was dried, autoradiographed, and
exposed to a PhosphorImager screen for 18 hours and analyzed using
ImageQuant software.
Effect of hypoxia on TGF- 1 mRNA levels were greater than mRNA levels of TGF- 2
or TGF- 3. The strongest effect of hypoxia was on TGF- 2 mRNA
levels, which increased by 4-fold after 4 hours of exposure to hypoxia
compared with untreated HUVECs, followed by 10-fold and 25-fold
increases at 24 hours and 48 hours, respectively (Figure 1A,B). At each
time point, mRNA from nonhypoxic control HUVECs was also analyzed, and
the TGF- mRNA profile from these cultures remained unchanged (data not shown). Viability of endothelial cells at 24 hours and 48 hours, as
disclosed by trypan blue and lactate dehydrogenase release, was more
than 95%. A strong effect of hypoxia was observed on TGF-
bioactivation (Figure 1C). The level of bioactive TGF- , which was
assayed in supernatants prior to activation of latent TGF- , was 12 times higher in supernatants from hypoxic HUVECs compared with normoxic
control supernatants. Thus, 64% of TGF- in supernatants from
hypoxic HUVECs was bioactive, while only 14% of total TGF- was
bioactive in the nonhypoxic supernatants20 (Figure 1C). The
level of total TGF- (which is determined after activation of latent
TGF- ) produced by HUVECs after 48 hours of exposure to hypoxia was
increased by 2.6-fold compared with nonhypoxic cells
(P < .05) both by the MLEC assay (Figure 1C) and by an
ELISA that specifically measures TGF- 2 (data not shown). As seen in
Figure 2, nonhypoxic HUVECs have readily
discernable levels of both TGF- RI and TGF- RII mRNA. While hypoxia
did not alter mRNA levels of TGF- RI, there was a modest but
consistent 1.8- to 2.5-fold increase in levels of the serine threonine
kinase TGF- RII in hypoxic HUVECs (P < .05) after
correction for L32 expression (data not shown), suggesting that in
these cells hypoxia could activate TGF- signaling as well.
Up-regulation of vascular endothelial growth factor in this experiment
(Figure 2) confirms hypoxic conditions.47
Level of hypoxia-induced TGF- 2 mRNA resulted from an up-regulation of its transcriptional activation, we used in vitro transcription assays performed with nuclei isolated from HUVECs exposed to 20% or 1% O2 for 24 hours. The results from 1 of 2 experiments that
yielded identical results are shown in Figure
3 and demonstrate that hypoxia increased
transcription of TGF- 2 by 15-fold after normalization to GAPDH (data
not shown). Similarly, transcription of the hypoxia-responsive glucose
transporter-1 (Glut-1)49 gene was also increased under hypoxic culture conditions, whereas transcription of the housekeeping gene GAPDH was largely unchanged. Furthermore, treatment with -amanitin to block type II RNA polymerase activity exhibited very
similar decay curves in hypoxic and normoxic HUVECs (Figure 4B), indicating that hypoxia's effect on
TGF- 2 gene expression occurred largely at the transcriptional
level.
To further study hypoxia-induced TGF-
TGF- We next examined whether hypoxia stimulated activity of another
promoter construct with known responsiveness to activation by Smad
proteins using a well described and widely used artificial promoter
construct, plasmid p3TP-Lux (Figure 6A),
which is responsive to TGF-
Nuclear extract binding to the Smad consensus sequence Smad binding to DNA was studied by comparing gel retardation patterns of nuclear proteins from HUVECs exposed to hypoxia, normoxia, or TGF- to DNA sequences that are specific for binding Smad
proteins. As the probe in the electrophoretic gel mobility shift assay,
we used the well-described oligonucleotide SBE which, as shown by x-ray
crystallography, can associate with Smad3 by either or both of its
palindromic Smad-binding sequences.29 As seen in Figure
7A, after exposure to hypoxia a discrete
gel-shifted band is observed that was absent from control nonhypoxic
nuclear extracts. This band was identical in position to the one
observed with nuclear extracts of TGF- 2-treated HUVECs and raised
the possibility that hypoxia induced Smad binding to DNA. Binding to
the SBE by nuclear extracts stimulated by hypoxia or by TGF- 2 was
competed by an unlabeled 37 bp oligonucleotide corresponding to the
hypoxia-responsive TGF- 2 promoter sequences between 77 and 40 bp
and containing a Smad-binding site. However, unlabeled oligonucleotide
corresponding to the HIF-1 binding site of the erythropoietin promoter
did not compete binding of hypoxic nuclear extracts to the
SBE. Results suggest that the shared Smad-binding site between
the SBE and the hypoxia-responsive TGF- 2 promoter between 77 and
40 bp can bind nuclear proteins in hypoxic but not normoxic cells and
that binding is independent of HIF-1.
To identify the transcription factors participating in the DNA
association in nuclear extracts from TGF-
In this report, we show that hypoxia increases TGF- Our results show that the HIF-1-binding oligonucleotide cannot compete
association of hypoxic nuclear extracts with SBE. In addition,
exposure to 200 µM hydrogen peroxide, which depletes HIF-1 Similar to our results, up-regulation of TGF- Nonuniform responses in key endothelial functions such as endothelial
cell growth, development, migration, and angiogenesis in response to
TGF- Our studies do not address the consequences of Smad activation and
TGF-
We are thankful to Dr Isil Aksan, Molecular Biology and Genetics Department, Bogazici University, Istanbul, for helpful discussions.
Submitted March 2, 2001; accepted July 19, 2001.
Supported by the American Heart Association Heritage Affiliate Grant-in-Aid (O.A.B.); National Heart, Lung, and Blood Institute grant HL53573 (M.S.); and National Institute of Mental Health grant MH599990 (E.S.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Olcay Ayanlar Batuman, Div of Hematology/Oncology, Dept of Medicine, SUNY Downstate Medical Center, Box 20, 450 Clarkson Ave, Brooklyn, NY 11203; e-mail: obatuman{at}downstate.edu.
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