(Received for publication, January 18, 1996, and in revised form, October 30, 1996)
From the Division of Pulmonary and Critical Care and
¶ Center for Medical Genetics, Departments of Medicine
and Pediatrics, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205 and
Department of Molecular
Genetics, Alton Ochsner Medical Foundation Medical Center,
New Orleans, Louisiana 70121
Exposure of rats to hypoxia (7% O2)
markedly increased the level of heme oxygenase-1 (HO-1) mRNA in
several tissues. Accumulation of HO-1 transcripts was also observed
after exposure of rat aortic vascular smooth muscle (VSM) cells to 1%
O2, and this induction was dependent on gene transcription.
Activation of the mouse HO-1 gene by all agents thus far tested is
mediated by two 5-enhancer sequences, SX2 and AB1, but neither
fragment was responsive to hypoxia in VSM cells.
Hypoxia-dependent induction of the chloramphenicol acetyltransferase (CAT) reporter gene was mediated by a 163-bp fragment
located approximately 9.5 kilobases upstream of the transcription start
site. This fragment contains two potential binding sites for
hypoxia-inducible factor 1 (HIF-1). A role for HIF-1 in HO-1 gene
regulation was established by the following observations: 1) HIF-1
specifically bound to an oligonucleotide spanning these sequences, 2)
mutation of these sequences abolished HIF-1 binding and
hypoxia-dependent gene activation in VSM cells, 3) hypoxia increased HIF-1
and HIF-1
protein levels in VSM cells, and 4) hypoxia-dependent HO-1 mRNA accumulation was not
observed in mutant hepatoma cells lacking HIF-1 DNA-binding activity.
Taken together, these data demonstrate that hypoxia induces HO-1
expression in animal tissues and cell cultures and implicate HIF-1 in
this response.
Low cellular oxygen tension is a feature of both physiological conditions, such as adaptations to high altitude and physical endurance exercise (1), and pathophysiological conditions including ischemia, fibrosis (2), and neoplasia (3). Mammalian cells respond to hypoxia in part by increased expression of several genes that encode both tissue-specific and ubiquitous proteins (4). These proteins participate in diverse biological processes including erythropoiesis, which enhances the oxygen carrying capacity of the blood; angiogenesis, which permits delivery of oxygen carrying blood to hypoxic sites; glycolysis, as a means of energy production; xenobiotic detoxification; and cellular adaptation to stress. Hypoxia-inducible proteins within these respective categories include erythropoietin (EPO)1 (5), vascular endothelial growth factor (6), glycolytic enzymes (7-9), NAD(P)H:quinone oxidoreductase (10), and heat shock proteins (11, 12). Where examined, increased expression of specific proteins in response to hypoxia is regulated primarily at the level of gene transcription (although post-transcriptional mechanisms have also been characterized).
Another stress-associated protein whose expression is stimulated by hypoxia is heme oxygenase-1 (HO-1) (13, 14). HO-1, a microsomal membrane enzyme, catalyzes the first and rate-limiting reaction in heme catabolism, the oxidative cleavage of b-type heme molecules to yield equimolar quantities of biliverdin, carbon monoxide (CO), and iron. Biliverdin is subsequently converted to bilirubin by the action of biliverdin reductase. The expression of HO-1 is dramatically induced not only by the substrate, heme, but a variety of stress-associated agents, including heavy metals, hyperthermia, and UV irradiation (reviewed in Maines (15)). A common feature among these inducers, including heme, is that they generate reactive oxygen species and/or diminish glutathione levels. This correlation and the observation that bilirubin functions as an antioxidant (16) has led to the hypothesis that induction of HO-1 is part of a general response to oxidant stress and that this enzyme plays a protective role during such conditions (17-19).
Stimulation of HO-1 expression by most if not all inducers is
controlled primarily at the level of gene transcription and in our
studies on the regulation of the mouse HO-1 gene, we have identified
two 5 distal enhancer regions, SX2 and AB1, that mediate gene
activation by a variety of pro-oxidants including heme, heavy metals,
TPA, hydrogen peroxide, and LPS (20-23). The mechanism of HO-1
induction by hypoxia has not been investigated and because this
induction has been proposed to occur as a consequence of oxidative
stress (13), we examined the role of the SX2 and AB1 enhancers in
hypoxia-dependent gene activation. In this report we show
that these enhancers do not mediate transcriptional activation of the
HO-1 gene in response to hypoxia. Rather, this induction is mediated by
a 163-bp fragment located directly downstream of the AB1 enhancer and
approximately 9.5 kb upstream of the transcription initiation site. The
active sequences within this fragment resemble the binding site for
hypoxia-inducible factor-1 (HIF-1), initially identified as a protein
that binds to a site in the EPO gene enhancer required for
transcription activation by hypoxia (24, 25). Additional data are
provided to support a role for HIF-1 in HO-1 gene regulation.
Pathogen-free Harlan Sprague Dawley rats (200-225 g) were purchased from Harlan Sprague Dawley (Indianapolis, IN) and allowed to acclimate upon arrival for 7 d prior to experimentation. Animals were fed rodent chow and water ad libitum. Animals were exposed to hypoxia (7.0% O2) in a 3.70-cubic feet plexiglass exposure chamber. Rats were supplied with rodent chow and water ad libitum during the exposures. At appropriate times, animal(s) were removed from the chamber and immediately sacrificed by decapitation. Tissues were removed and frozen in liquid nitrogen for subsequent RNA extraction. These experiments were carried out according to animal protocols approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine.
Cell CulturePrimary cultures of rat aortic vascular smooth
muscle (VSM) cells, passage 2-10, were generously provided by Dr.
Michael Crow of the National Institute of Aging. Chinese hamster ovary
epithelial cells (CHO cells) were obtained from American Tissue Cell
Culture. VSM cells were maintained in Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum and gentamicin (50 µg/ml). CHO cells were maintained in Ham's-F12 medium. Wild type
Hepa1 c1c7 and mutant Hepa1 c4 cells (generously provided by Dr. O. Hankinson of the University of California at Los Angeles) were maintained in minimal essential medium- supplemented with 10% fetal
bovine serum. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2, 95% air. Cells were exposed to
hypoxia (1% O2, 5% CO2, balance
N2) in a tightly sealed modular incubator chamber
(Billups-Rothberg, Del Mar, CA) at 37 °C. All experimentations were
performed with confluent cultures.
Total RNA from
cells and tissues was isolated by the acid guanidinium
thiocyanate-phenol-chloroform extraction method (26). Northern blot
analyses were performed as described previously (23). Briefly, 10- or
15-µg (Hepa1 cells) aliquots of total RNA were fractionated on a
denaturing agarose gel, transferred to nylon membrane by capillary
action, and cross-linked to the membrane by UV irradiation. The nylon
membranes were incubated in hybridization buffer (1% bovine serum
albumin, 7% SDS, 0.5 M phosphate buffer, pH 7.0, 1.0 mM EDTA) containing 32P-labeled rat HO-1
cDNA (27) (generously provided by Dr. S. Shibahara of Tohuku
University, Japan) at 65 °C for 24 h. Nylon membranes were then
washed twice in buffer A (0.5% bovine serum albumin, 5% SDS, 40 mM phosphate buffer, pH 7.0, 1 mM EDTA) for 15 min at 65 °C followed by four washes in buffer B (1% SDS, 40 mM phosphate buffer, pH 7.0, 1.0 mM EDTA) for
15 min at 65 °C and exposed to x-ray film. To control for variation
in either the amount of RNA in different samples or loading errors,
blots were hybridized with a radiolabeled oligonucleotide probe
(5-ACGGTATCTGATCGTCTTCGAACC-3
) complementary to 18 S rRNA after
stripping of the HO-1 probe. Autoradiographic signals were quantified
by densitometric scanning (Molecular Dynamics, Sunnyvale, CA). All
densitometric values obtained for the HO-1 mRNA transcript (1.8 kb)
were normalized to values for 18 S rRNA obtained on the same blot. The
HO-1 mRNA level in treated cells was expressed in densitometric
absorbance units, normalized to control untreated samples, and
expressed as fold induction compared to controls.
For HO-1 immunoblots, frozen tissues or cells were homogenized in lysis buffer (1% Nonidet P-40, 20 mM Tris, pH 8.0, 137.5 mM NaCl, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin). Protein concentrations of the lysates were determined by Coomassie Blue dye-binding assay (Bio-Rad). An equal volume of 2 × SDS/sample buffer (0.125 M Tris-HCl, pH 7.4, 4% SDS, and 20% glycerol) was added and the samples were boiled for 5 min. Samples (100 µg) were subjected to electrophoresis in a 12% SDS-polyacrylamide gel and then transferred electrophoretically onto a polyvinylidene fluoride membrane. The membranes were incubated for 2 h with rabbit polyclonal antibody against rat HO-1 (1:1,000 dilution; StressGen Biotech Corp., Vancouver, Canada) followed by incubation with goat anti-rabbit IgG antibody for 2 h. Signal development was carried out using an ECL detection kit (Amersham Corp.).
For HIF-1 and HIF-1
immunoblots, aliquots of crude nuclear
extracts containing 15 µg of protein from cells exposed to 1% or
20% O2 were fractionated at 30 mA by SDS-PAGE using a 7%
acrylamide, 0.23% bisacrylamide gel. The proteins were transferred to
nitrocellulose membranes, incubated with affinity-purified HIF-1
or
HIF-1
antibodies at 1:600 (v/v) dilution. The membrane was washed,
incubated with horseradish peroxidase anti-immunoglobulin conjugate at
1:2000 dilution, and detected with ECL reagents as described previously (28).
Construction of
the CAT reporter plasmids diagramed in Fig. 4 has been described
previously (20-22). Subfragments (see Fig. 5) of fragment EH (formerly
EH2) (22) were isolated after digestion with the appropriate
endonucleases, blunt-ended, and cloned into the SpeI site,
upstream of the minimal promoter and CAT gene in pMHO1CAT-33 (20).
Mutation of the putative HIF-1 binding sites in the BT fragment was
carried out by oligonucleotide-directed mutagenesis (29) using the
single-stranded form of plasmid pMHO1CAT
-33 + BT. The sequence of
the mutagenic oligonucleotide was
5
-CGCTCTAGAACTAGCGGAAAGCTGGCGTGGCTTTTCCTCTCTG-3
. The first 14 residues are derived from the multiple cloning sites of pBluescript II
SK
, the parent plasmid of pMHO1CAT
-33. Residue 15 corresponds to
nucleotide 324 in the sequence presented in Fig. 6A.
Plasmids (10 µg) were transiently transfected into cells using Lipofectin Reagent (Life Technologies, Inc.) according to the manufacturer's protocol. The cells were transfected overnight after which time the plates were washed twice with serum-free media and then incubated in complete media at 1 or 20% O2 for 24 h. Preparation of cellular extracts and measurement of CAT activity were carried out as described previously (30). CAT enzyme standard was purchased from Sigma and used for calculation of cellular enzyme activities.
Electrophoretic Mobility Shift AssayRat VSM cells were
exposed to 20% or 1% O2 for 6 h and crude nuclear
extracts were prepared as described previously (24). Electrophoretic
mobility shift assay using 7 µg of nuclear extracts was performed in
binding buffer containing 25 mM Tris-HCl (pH 7.6), 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, and 1.2 mM sodium vanadate, with 104
cpm of oligonucleotide probe (24, 31). Oligonucleotide competition experiments were performed with 1 or 3 ng (10- or 30-fold excess) of
unlabeled wild-type or mutant HO-1, or 3 ng of unlabeled wild-type or
mutant EPO double-stranded oligonucleotides. After preincubation for 5 min at room temperature, labeled wild-type HO-1 probe was added and
incubated on ice for 15 min. In the supershift assays, 1 µl of
preimmune serum or antiserum (28) specific for HIF-1 at 1:3 dilution
or HIF-1
at 1:6 dilution was added to the reaction mixture and
incubated for 30 min on ice prior to electrophoresis.
Rats were
exposed to 7% O2 for 0, 0.5, 1, or 2 h and HO-1
expression in various tissues was monitored by RNA blot analysis. Results from a representative experiment are shown in Fig.
1. A marked increase in the steady-state level of HO-1
mRNA was observed in the lung, liver, heart, and aorta, and in all
cases this induction was evident within 30-60 min of exposure to
hypoxia. Within the time period examined, the highest levels of
induction were observed in the liver (32-fold) and lung (16-fold).
Reexposure of rats to 21% O2 for 15 or 30 min after 2 h of hypoxia resulted in a rapid decline in the amount of HO-1 mRNA
in all tissues examined. HO-1 mRNA accumulation was also observed
in the kidney and adrenal gland but was less pronounced. No induction
was observed in the spleen (data not shown).
Induction of HO-1 Expression in VSM Cells Subjected to Hypoxia
Previous studies have demonstrated
hypoxic-dependent induction of HO-1 expression in CHO cells
(13) and VSM cells (14). The latter were selected for investigation of
the regulatory mechanism of HO-1 induction because of the physiological
relevance of VSM cells (see below) and because they exhibited more
consistent and higher levels of induction than CHO cells (data not
shown). Exposure of VSM cells to 1% O2 produced an initial
rise in the steady-state amount of HO-1 mRNA at 4 h
(4.0-fold), peak levels at 8 h (6.0-fold), prior to a drop in the
levels by 24 h (2.4-fold) and 48 h (1.6-fold) of continuous
hypoxia (Fig. 2A). The level of HO-1 mRNA
accumulation and the temporal profile are similar to those observed
after exposure of VSM cells to 0% O2 (14). Induction of
HO-1 expression in cultured VSM cells appears to be delayed relative to
that observed in the aorta. This variation, however, is not surprising
given the differences in the treatment conditions and the cellular
environments, particularly the presence of multiple cell types
(i.e. endothelial cells) in the in vivo studies.
The accumulation of HO-1 mRNA in cultured VSM cells correlated with
increased levels of HO-1 protein (Fig. 2B).
To further delineate the molecular basis for increased expression of
HO-1 in response to hypoxia, we examined whether HO-1 mRNA
induction was dependent on gene transcription. Cells were pretreated
for 1 h with actinomycin D, a potent inhibitor of RNA synthesis,
prior to exposure to hypoxia. As shown in Fig. 3,
actinomycin D (1 µg/ml) completely inhibited hypoxia-induced HO-1
mRNA accumulation.
Identification of the Hypoxia-responsive Elements in the Mouse HO-1 Gene
To identify cis-acting DNA sequences that mediate
HO-1 gene activation in response to hypoxia, VSM cells were transiently transfected with plasmids containing various segments of the
5-flanking region of the mouse HO-1 gene linked to the
Escherichia coli CAT gene. Transfected cells were exposed to
20% O2 or 1% O2 for 24 h and
hypoxia-dependent fusion gene regulation was assessed by measuring CAT activity in cellular extracts. Analysis of approximately 12.5 kb of the HO-1 5
-flanking region indicated that the
hypoxia-responsive element (HypRE) resides within a 900-bp fragment,
EH, located 9 kb upstream of the transcription initiation site (Fig.
4). By analyzing subfragments of EH in a manner
analogous to that described above, the HypRE was localized to a 163-bp
BsrBI/TaqI fragment, BT (Fig. 5).
Interestingly, this fragment is immediately downstream of enhancer
sequences, AB1, previously shown to mediate transcriptional activation
of the HO-1 gene in response to various agents including heme and
cadmium (22). Neither the AB1 enhancer nor the SX2 enhancer (Fig. 4)
were responsive to hypoxia in VSM cells.
A partial sequence of the EH fragment, is shown in Fig.
6A. The sequence of the AB1 segment (residues
166-326) has been published previously (22). Computer analysis of the
EH sequence indicated that it contains two motifs similar to the
consensus binding site, 5-BACGTGCK-3
, for HIF-1 (9). Both of the
putative HIF-1 binding sites 5
-GACGTGCT-3
(sense strand) and
5
-GACGTGCC-3
(antisense strand) are located within the
hypoxia-responsive BT fragment (residues 324-486). To assess the role
of these putative HIF-1 binding sites in hypoxia-dependent
gene activation, the CGT trinucleotide of both elements was mutated to
AAA in the context of the BT fragment (Fig. 6B). Previous
studies have demonstrated that analogous mutations within the HIF-1
binding site of the hypoxia-responsive EPO enhancer, 5
-TACGTGCT-3
,
abolish both protein binding and enhancer function (24, 32) (see
below). The mutant BT fragment was unresponsive to hypoxia (Fig.
6B), demonstrating that these sequences are necessary for
HO-1 gene activation in hypoxic VSM cells.
Double-stranded oligonucleotides were synthesized
corresponding to wild-type and mutant HO-1 and EPO sequences (Fig.
7A). The wild-type HO-1 oligonucleotide
(Hw) was radiolabeled and incubated with nuclear extracts
prepared from VSM cells that had been incubated in 1% or 20%
O2 for 6 h. The Hw probe detected a
constitutively-expressed DNA-binding activity (C) that was
present in both nonhypoxic (Fig. 7B, lane 1) and
hypoxic (lane 2) cells as well as a DNA binding activity
present only in hypoxic cells (lane 2). Inclusion of 1 or 3 ng (representing a 10- or 30-fold molar excess) of unlabeled mutant
HO-1 oligonucleotide (Hm) had no effect on the binding of
the inducible factor(s) to the Hw probe (lanes 3 and
4), whereas binding was partially and completely inhibited
by 1 and 3 ng, respectively, of unlabeled Hw oligonucleotide
(lanes 5 and 6). Binding of the inducible
factor(s) to the Hw probe was also blocked by excess wild-type
(Ew), but not mutant (Em), EPO oligonucleotides (lanes 7 and 8). Inclusion of antiserum raised
against HIF-1 (Fig. 7C, lane 2) or HIF-1
(lane 4) resulted in the loss of the inducible complex and
formation of a supershifted complex, suggesting that HIF-1
and
HIF-1
are components of the hypoxia-inducible HypRE binding
activity. The respective preimmune sera (lanes 1 and
3) had no effect on the HIF-1/DNA complex that formed in the absence of antiserum (lane 5).
HIF-1 and HIF-1
protein levels in nuclear extracts from
non-hypoxic and hypoxic VSM cells were also quantified by immunoblot assay (Fig. 8). Levels of HIF-1
were extremely low in
nonhypoxic cells and increased dramatically in response to hypoxia.
Multiple HIF-1
isoforms were detected in hypoxic VSM cells. Compared
to HIF-1
, HIF-1
protein levels were higher in nonhypoxic VSM
cells and showed a more modest induction in response to hypoxia. These features of HIF-1 expression are similar to those previously described for Hep3B cells (28). Taken together, these results demonstrate that
(i) exposure of VSM cells to 1% O2 increases nuclear HIF-1 DNA-binding activity as well as HIF-1
and HIF-1
protein, (ii) HIF-1 specifically binds to HO-1 sequences necessary for gene activation in response to hypoxia, and (iii) mutations in the HO-1
enhancer that result in a loss of transcriptional activity in hypoxic
cells also result in a loss of HIF-1 binding.
HO-1 mRNA Is Not Induced by Hypoxia in Cells Lacking HIF-1 DNA Binding Activity
To further investigate the role of HIF-1 in HO-1
gene regulation, two clones of mouse hepatoma cells, wild-type Hepa1
c1c7 and its mutant derivative Hepa1 c4, were analyzed for HO-1
expression under hypoxic conditions. Hepa1 c4 cells are deficient in
HIF-1 (33) and lack HIF-1 DNA-binding
activity.2 These cells were incubated for
0, 4, 8, and 16 h at 1% O2, and HO-1 expression was
subsequently assessed by Northern blot analyses. In wild-type Hepa1
cells, HO-1 mRNA levels increased 14.0-fold at 4 h, 7.8-fold
at 8 h, and 4.3-fold at 16 h of hypoxia (Fig. 9). In mutant Hepa1 c4 cells, the basal level of HO-1
mRNA was significantly higher but no appreciable increase was
observed under hypoxic conditions. These data provide further evidence that HIF-1 plays a critical role in HO-1 gene activation in response to
hypoxia in cultured cells.
Much interest in HO-1 has been generated recently by reports demonstrating the induction of HO-1 expression by a variety of pro-oxidants including UV irradiation, hyperoxia, and endotoxin, and the protection this induction affords against heme- and non-heme-mediated oxidant injury (reviewed in Choi and Alam (34)). The present study demonstrates that acute hypoxic stress also stimulates HO-1 expression both in animal tissues and in cell cultures. In VSM cells, induction of HO-1 expression is regulated at the level of gene transcription and results from several lines of investigation demonstrate that this activation is mediated by HIF-1.
HIF-1 was initially identified as a hypoxia-inducible nuclear factor in
Hep3B hepatoma cells that bound to sequences necessary for hypoxic
activation of the EPO gene, whose product is expressed primarily in
fetal liver and the kidney. Subsequent studies demonstrated that HIF-1
is present and/or functions in cell lines of various tissue origin,
including HeLa cervical carcinoma cells, Ltk fibroblast,
C2C12 skeletal myoblasts and CHO cells,
suggesting that this transcription factor plays a more general role in
the hypoxic response of mammalian cells and may regulate the expression of other hypoxia-inducible genes (32). Indeed, HIF-1 binding sites have
been identified in other functional classes of hypoxia-inducible genes
including those coding for inducible nitric oxide synthase (35),
vascular endothelial growth factor2 (36, 37), and the
glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, and
phosphoglycerate kinase 1 (7, 9). Given the potential significance of
increased HO-1 activity in hypoxic cells (see below), the
identification of the HO-1 gene as a target for HIF-1 regulation lends
further support to the hypothesis that HIF-1 coordinates homeostatic
transcriptional responses to cellular hypoxia.
In addition to HIF-1, the AP-1 transcription factors, comprised of homo- and heterodimers of the Fos and Jun family of proteins, have been implicated in hypoxia-dependent gene activation. For instance, in several cell types, both the mRNA levels of individual Fos and Jun family members and AP-1- specific DNA binding activity are increased after exposure to hypoxia (10, 38, 39). Furthermore, an AP-1 binding site within the promoter of the tyrosine hydroxylase gene is required for hypoxia-dependent transcription activation (40). Both SX2 and AB1 enhancers contain binding sites for various transcription factors, including CCAAT/enhancer binding protein and Sp1, but the dominant element within both fragments is the consensus sequence, (T/C)GCTGAGTCA, which is present in five copies (20, 22). Mutation of these elements, which are targets for various AP-1 member proteins (20) (data not shown), dramatically diminishes or completely abolishes gene activation by all SX2- and AB1-dependent inducers thus far tested including heme, heavy metals, hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate, and lipopolysaccharide (22, 23, 30, 41). That a single type of element regulates mouse HO-1 induction by diverse agents argues for a common mechanism in the mode of action of these agents and is consistent with the oxidant stress hypothesis. Clearly, neither the SX2 nor the AB1 enhancer is responsive to hypoxia in VSM cells. These results attest to the uniqueness of hypoxia as an inducer of the mouse HO-1 gene and argue against the previous suggestion that hypoxic induction of HO-1 in CHO cells occurs as a consequence of oxidative stress (13).
The significance of increased HO-1 expression under hypoxic conditions is not clearly understood. The utilization of distinct transcription factor pathways for HO-1 gene activation by hypoxia and various pro-oxidants, however, suggests that this significance may be less related to the antioxidant activity of HO-1 (i.e. production of bilirubin) than to the ability of this enzyme to generate CO. CO is a member of a new class of gaseous signaling molecules that also includes nitric oxide (NO). CO, like NO, can activate guanylate cyclase resulting in elevated levels of cGMP, which among other activities, causes relaxation of VSM cells and inhibition of platelet aggregation. Recent studies have in fact established that, under physiological conditions, maintenance of normal vascular tone and blood flow can be attributed in part to enzymatically derived CO (42, 43). Therefore, changes in vascular tone, endothelial permeability, and/or coagulant function observed under hypoxic conditions (44, 45) may in part be a consequence of HO-1 induction and localized production of CO. Consistent with this role of HO-1, hypoxic VSM cells exhibit increased production of CO, derived primarily if not exclusively from induced HO-1 activity, and increased levels of cGMP (14). Furthermore, the HO-1-derived CO from VSM cells may have paracrine effects as it modulates cGMP levels and gene expression in endothelial cells in an in vitro co-culture system (46). Finally, with respect to VSM cells, HO-1 induction and the regulatory effects of CO may be of particular significance in pathophysiological conditions such as atherosclerosis where smooth muscle cells are exposed to hypoxic environments prior to and during development of lesions (47, 48).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U70472[GenBank].
We thank Cara Zbylut for assisting in preparation of the manuscript.