Research Paper
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Acta Biochim Biophys Sin
2005,37:665–672
doi:10.1111/j.1745-7270.2005.00095.x
Differential Expression of Three Hypoxia-inducible Factor-a Subunits in
Pulmonary Arteries of Rat with Hypoxia-induced Hypertension
Qi-Fang LI& and Ai-Guo DAI*
Department of
Respiratory Medicine, Hunan Institute of Gerontology, Hunan Province Geriatric
Hospital, Changsha 410001, China
Received: June 7,
2005
Accepted: July 27,
2005
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30270581), the Major Foundation of Hunan Province Educational Committee (No.
02A047), the Postdoctorate Science Foundation of China (No. 2003033436) and the
Major Science and Technology Foundation of Ministry of Education (No. 03091)
& Present address:
Department of Anesthesiology, Renji Hospital, Shanghai Second Medical
University, Shanghai, China
*Corresponding
author: Tel, 86-731-4762793; Fax, 86-731-4735215; E-mail, [email protected]
Abstract Hypoxia inducible transcription
factor (HIF)-1a plays an important role in the development of hypoxic pulmonary hypertension,
but little is known about HIF-2a and HIF-3a with respect to
transcriptional regulation by hypoxia. To examine the expression patterns
of all HIF-a subunits (HIF-1a, HIF-2a and HIF-3a) in pulmonary arteries of rats undergoing systemic hypoxia, five groups
of healthy male Wistar rats were exposed to normoxia (N) and hypoxia for 3
(H3), 7 (H7), 14 (H14) and 21 (H21) d respectively. Mean pulmonary arterial
pressure (mPAP), vessel morphometry and right ventricular hypertrophy
index were measured. Lungs were inflation fixed for immunohistochemistry and
in situ hybridization, and homogenized for Western blot. mPAP
increased significantly after 7 d of hypoxia [(18.4±0.4) vs. (14.4±0.4)
mmHg, H7 vs. N], reached
its peak after 14 d of hypoxia, then remained stable. Pulmonary artery remodeling
and right ventricular hypertrophy developed significantly after 14 d of hypoxia.
During normoxia, HIF-1a and HIF-3a staining were slightly positive
regarding mRNA levels. A substantial alteration of HIF-1a and HIF-3a staining
occurred in pulmonary arteries after 14 d and 7 d of hypoxia, respectively,
but HIF-2a staining showed an inversed trend after 14 d of hypoxia. Protein levels
of all HIF-a subunits except HIF-3a showed a marked increase corresponding to the
duration of hypoxia, which was obtained by Western blot. Our study found that
HIF-1a, HIF-2a and HIF-3a may not only confer different target genes, but also play key pathogenetic
roles in hypoxic-induced pulmonary hypertension.
Key words hypoxia inducible
factor-a; hypoxia; hypertension; lung
Acute hypoxia constricts reversibly the pulmonary arteries and
dilates systemic arteries, a process known as hypoxia pulmonary
vasoconstriction. Chronic hypoxia leads to structural remodeling of the
vessels, comprising increased thickness of the adventitial and medial layers,
and perhaps more importantly, the muscularization of precapillary vessels that
either have poor muscularization or are devoid of muscle under normal
conditions. The combination of vasoconstriction and vascular remodeling, as
well as an increase in hematocrit, result in pulmonary hypertension and,
subsequently, right ventricular hypertrophy [1,2]. Hypoxia inducible
transcription factor-1 (HIF-1) gene and its dependent target genes, with the
hypoxia-responsive element as the regulatory component, are strongly activated
in chronic hypoxia [3]. HIF-1 is a heterodimeric bHLH transcription factor that
consists of an oxygen-regulated functional a-subunit, and a
constitutively expressed b-subunit also known as aryl hydrocarbon receptor nuclear
translocator (ARNT). HIF-1 mediates complex cellular and systemic adaptive responses
to a reduced oxygen supply, stimulating transcriptions of a large array of
glycolytic and vasoactive genes, iron metabolism genes, growth factor genes, erythropoietin
genes and many angiogenic factor genes [4,5]. Besides the well-established
HIF-1a, two other members of the bHLH-PAS superfamily have also been
described: HIF-2a [6] and HIF-3a [7], which bear similar functions to HIF-1a regarding hypoxic
stabilization and binding to ARNT. The role of HIF-1a in cellular response to
hypoxia is well established, whereas little is known about HIF-2a and HIF-3a with respect to
dynamic expression in pulmonary arteries during chronic hypoxia.To unravel the roles of HIF-2a and HIF-3a, and the
susceptibility of HIF-a subunits to chronic hypoxic conditions, we studied the expression
patterns of HIF-1a, HIF-2a and HIF-3a in pulmonary arteries of rats at different phases of
hypoxia-induced pulmonary hypertension development.
Materials and Methods
Materials
Hypoxia and normoxia rat models were set up as described previously
[8]. The hypoxic condition was established by flushing the chamber intermittently
with a gas mixture of room air and nitrogen from a liquid nitrogen reservoir.
An HT-6101 oxygen analyzer (Kangda Electrical Company Limited, Chengdu,
China) was used to monitor the chamber environment (the chamber was ventilated
by a hole, then a dynamic balance was achieved through the inspiration and
expiration of the rats). CO2 was removed with
soda lime, excess humidity was prevented by anhydrous calcium chloride,
and ammonia was kept to a minimum level by boric acid in the chamber. For
hypoxia models, 40 Wistar rats (male, 220±10 g, 6–8 weeks old) from the Animal
Experimental Center of Center South University (Changsha, China) were randomly
divided into five groups, with eight rats in each group. Hypoxic rats were
exposed to normobaric hypoxia at (10.0±0.5)% O2 for
3 (H3), 7 (H7), 14 (H14) and 21 (H21) d (8 h per day, intermittently), respectively,
in a ventilated chamber. The control rats were kept in a normoxic ventilated
chamber (21% O2) in the same chamber, and were killed after
being caged for 10 d, because breeding duration has no significant
effect on mean pulmonary arterial pressure (mPAP), hypoxic pulmonary
artery remodeling or right ventricular hypertrophy index (RVHI) [8].
mPAP and RVHI measurements
mPAP was measured as described
previously [9]. Rats were intraperitoneally anesthetized with 40 mg
pentobarbital sodium per 1 kg body weight. A specially designed single-lumen
catheter was then inserted into the main pulmonary artery through the right
jugular vein; the injecting position was decided through the waveform of
pressure. Through this catheter, mPAP was measured using PowerLab monitoring
equipment (AD Instruments Pty Limited, Milford, USA). For RVHI
measurement, each heart was cut open and atria were removed. The right
ventricular free wall was dissected, and each chamber weighed. Equation 1
was used to calculate the RVHI, in which RV is the weight of the
right ventricle, LV is the weight of the left ventricle, and S is
the weight of the septum.
1
Morphometric analysis
Lung slices of 5 mm were embedded with paraffin, stained with hematoxylin and eosin,
followed by elastic fiber staining, then examined with light microscopy. At least
five representative pulmonary arteries (100–150 mm in outer
diameter) chosen from three different sections from each animal were
independently examined. To evaluate hypoxic remodeling by calculating the
parameters of pulmonary vascular cross-sections, the ratio of vascular wall
area to total vascular area (WA) and pulmonary artery media thickness (PAMT)
were measured. The images of the arteries were captured and analyzed using
PIPS-2020 image software (Chongqing Tianhai Company, Chongqing, China).
In situ hybridization of HIF-1a, HIF-2a
and HIF-3a
In situ hybridization was performed
using an In situ hybridization detection kit (Wuhan Boster Biological Technology
Company, Wuhan, China). The oligonucleotide probes were designed by Boster
according to the sequences of rat HIF-1a, HIF-2a and HIF-3a.
The sequences of probes against HIF-1a mRNA were: (1) 5‘-TTATGAGCTTGCTCATCAGTTGCCACTTCC-3‘; (2) 5‘-CTCAGTTTGAACTAACTGGACACAGTGTGT-3‘; (3) 5‘-GGCCGCTCAATTTATGAATATTATCATGCT-3‘. The sequences of probes against HIF-2a mRNA were: (1) 5‘-CGAACACATAAACTCCTGTCTTCAGTGTGC-3‘;(2) 5‘-ATCCGAGAGAACCTGACACTCAAAACTGGC-3‘; (3) 5‘-GGGCAAGTGAGAGTCTACAACAACTGCCCC-3‘. The sequences of probes against HIF-3a mRNA were:(1) 5‘-CGCATGCACCGCCTCTGCGCTGCAGGGGAG-3‘; (2) 5‘-ACATGGCTTACCTGTCGGAAAATGTCAGCA-3‘; (3) 5‘-ATATGAGGGCCTACAAGCCCCCTGCACAGA-3‘.
Hybridization was performed on
serial lung tissue slices in paraffin fixed by formalin containing 0.1% diethypyrocarbonate
according to the manufacturers instructions. Briefly, slices were digested
with pepsin for 20 min at 37 ?C. After 2 h of pre-hybridization, slices
underwent hybridization with digoxin-labeled single-stranded oligonucleotide
probes for 16 h at 38 ?C. In negative control studies, labeled oligonucleotide
probes were substituted by phosphate-buffered saline (PBS). After washing off
unbound probes, slices were incubated first with rabbit antibodies against
digoxin, then with biotinylated goat antibodies against rabbit. Slices were
then incubated with SABC-peroxidase. Peroxidase activity was visualized by a
color reaction using diaminobenzidine (Boster) as the substrate. Brown and
yellow colors indicated positive results (mainly in cytoplasm). Finally, the
sections were counterstained with hematoxylin (resulting in blue nuclei) and
mounted.
Immunohistochemistry for HIF-1a, HIF-2a
and HIF-3a
A commercial SABC kit (Boster) was used for immunohistochemistry, which
was performed similar to that described previously with minor modification [3].
Briefly, serial sections of formalin-fixed paraffin-embedded lung tissues were
digested with 3% H2O2 for 20 min at room temperature, then preincubated with 10%
non-immunized serum. Sections were incubated with rabbit anti-HIF-1a (Boster),
anti-HIF-2a and anti-HIF-3a (Santa Cruz Biotechnology, Heidelberg, Germany) specific polyclonal
antibodies (1:150) overnight at 4 ?C. In negative control studies, the
antibodies were substituted by PBS. After unbound antibodies were washed off,
the sections were incubated with corresponding biotinylated second antibodies
against rabbit and thereafter incubated with streptavidin peroxidase.
Subsequently, peroxidase activity was visualized by a color reaction with
diaminobenzidine similar to that of in situ hybridization.
Western blot analysis for HIF-1a, HIF-2a and HIF-3a in lung tissue
Proteins from whole tissue samples were extracted as described
previously [10] using a modified homogenization buffer containing 10 mM Tris
(pH 8.0), 1 mM EDTA, 400 mM NaCl, 0.1% igepal CA-630, 1 mM dithiothreitol, 1 mM
phenylmethylsulphonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin
(Sigma, Deutschland, Germany). Protein concentrations were determined by the
Lowry method (Merck, Darmstadt, Germany). Equal amount of proteins were
separated in 7.5% sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE), transferred onto a polyvinylidene difluoride membrane (Millipore,
Eschborn, Germany) and stained by Coomassie blue. Detection of the target
proteins was accomplished using specific antibodies of HIF-2a, HIF-3a, b-actin (Santa
Cruz), or HIF-1a. Immunocomplexes were then labeled with peroxidase-conjugated
anti-mouse or anti-rabbit IgG (Boster). An enhanced chemiluminescence
detection kit (Amersham, Buckinghamshire, UK) was used for signal detection. Fotolook and NIH
image software programs were used for quantification.
Statistical analysis
Data are expressed as mean±SD. One-way analysis of variance
(ANOVA) was used to determine statistically significant difference in
more than two groups, and Newman-Keuls test was used to analyze statistical
significance between two groups. P<0.05 was considered as a statistically significant difference.
Results
mPAP increase induced by chronic hypoxia
mPAP was measured as an indicator of pulmonary
artery pressure in conscious rats. In normoxic rats, mPAP was
14.4±0.4 mmHg. In hypoxic animals, mPAP changed as expected:
the pulmonary hypertension was increased after 7 d exposed to hypoxia
(P<0.05), reached its peak after 14 d of hypoxia, then remained stable as the hypoxia condition was prolonged (Table 1).
Chronic hypoxia induced hypoxic pulmonary vascular remodeling and
right ventricular hypertrophy
Increased thickness of pulmonary arteries (PAMT and WA)
due to smooth muscle cell hypertrophy and hyperplasia is the structural
hallmark of pulmonary hypertension. As shown in Table 1, pulmonary arteries
in normoxic animals were thin, whereas after 7 d of hypoxic exposure they
developed increased medial thickness characteristic of pulmonary hypertension.
Quantification of these structural changes in several lung slices from all
three hypoxia groups (H7, H14 and H21)
revealed significantly increased medial thickness of pulmonary arteries in
hypoxic animals compared with normoxic controls. Right ventricular hypertrophy
is a hallmark of pulmonary hypertension resulting from right ventricle pressure
overload. After 14 d of hypoxia, RVHI was significantly increased
compared with the control value, and increased further after 21 d of hypoxia.
These data indicated that right ventricular hypertrophy was developed after 14
d exposed to hypoxia (Table 1).
mRNA level of HIF-1a, HIF-2a and HIF-3a in pulmonary arterial wall during normoxia and chronic hypoxia
conditions
To determine dynamic changes at the transcriptional level, the mRNAs
of HIF-1a, HIF-2a and HIF-3a were monitored during normoxia and after different hypoxic periods.
The changes of the HIF a-subunits were quite different to each other in the investigated
arteries (Figs. 1–3). In all arteries, distinct mRNA
staining of HIF-1a (Fig. 1) and HIF-3a (Fig. 3) were detected after 14 d and
7 d of hypoxia respectively; they were faintly traceable during normoxia, even
after 3 d of hypoxia. In contrast to HIF-1a and HIF-3a, HIF-2a presented a
completely different expression pattern. mRNA staining of HIF-2a lessened
drastically after 14 d of hypoxia, whereas strong HIF-2a mRNA staining was detected
during normoxia and after 3 d and 7 d of hypoxia (Fig. 2). Notably, with
regard to time periods, HIF-2a mRNA staining lessened while HIF-3a mRNA was
strongly stained. In summary, moderate O2 concentration
induced significant changes in the mRNAs of HIF-1a, HIF-2a and HIF-3a.
Protein level of HIF-1a, HIF-2a and HIF-3a in pulmonary
arterial wall and lung tissue during normoxia and chronic hypoxia conditions
In immunohistochemical analysis, antibodies against HIF-1a, HIF-2a and HIF-3a produced
different signals respectively (Figs. 4–6). All HIF-a protein levels enhanced gradually during hypoxia, but the
expression pattern differs to each other, implying the specific stabilization
of HIF-a protein by hypoxia. All HIF-a stainings were slightly positive
in control rats. But staining of HIF-1a was positive after 3 d and 7 d of hypoxia,
then lessened (Fig. 4). HIF-2a was stained strongly after 14 d of hypoxia,
then remained unchanged (Fig. 5). HIF-3a staining became strong
after 7 d of hypoxia, and remained stable thereafter (Fig. 6). In Western blot analysis, interestingly, even in normoxia, all HIF-a proteins except
HIF-3a in the lung tissue could be clearly detected, but HIF-1a and HIF-2a proteins enhanced
gradually after 3 d of systemic hypoxia and increased further as the duration
of hypoxia prolonged (Fig. 7), indicating the stabilization of the two
proteins by hypoxia.
Discussion
The main finding of this study is that the expression patterns of
HIF-1a, HIF-2a and HIF-3a are quite different during hypoxic pulmonary hypertension
development. Chronic hypoxia induced mPAP increases pulmonary vascular
muscularization and right ventricular hypertrophy. Importantly, we are able to
document the dynamic expression of HIF-2a and HIF-3a during
prolonged hypoxia. These data show that HIF-1a, HIF-2a and HIF-3a, which may
confer different target genes, also play key pathogenetic roles in the
mechanisms of hypoxic pulmonary hypertension.Previously we have shown that HIF-1a is one of the pivotal
mediators in the pathogenesis of hypoxia-induced pulmonary hypertension
development in rat, and most presumably through target genes such as the
inducible nitric oxide synthase gene, vascular endothelial growth factor gene
and heme oxygenase-1 gene [8,9]. In this study, the rise in pulmonary artery
pressure was most pronounced during the first week of hypoxia and reached a
plateau after 14 d, with only a small further increase during prolonged
hypoxia until 21 d. The sustained elevation in pulmonary artery pressure in
chronic hypoxia results, for the most part, from architectural changes in the pulmonary
vascular bed as reflected by the significant increase in WA and PAMT.
Pulmonary vascular remodeling in chronic hypoxia has many features of injury
repair. Accumulation of vascular smooth muscle has long been recognized as a
typical feature of hypoxic pulmonary vascular remodeling [11]. As homologs to
HIF-1a, there are very limited data about the expression of the HIF-2a and HIF-3a subunits at the
levels of mRNA and the protein in pulmonary arteries during normoxic and
hypoxic conditions. In one earlier study, specific mRNAs of HIF-1a, HIF-2a and HIF-3a were clearly
detectable by real-time reverse transcription (RT)-PCR in rat lung under
normoxic and acute hypoxic conditions [10]. In this study, the staining of HIF-1a and HIF-2a mRNA were
not affected by acute hypoxia (7 d of hypoxia) in pulmonary arteries. Regarding
HIF-1a, this is in line with earlier suggestions that other factors except
hypoxia may increase the expression of HIF-1a mRNA. In that study,
at no time point did an increase in HIF-1a appear, even after 12 h of
severe hypoxia [12]. Another study on rat brain revealed by Northern blot
analysis that an increase in HIF-1a could be achieved only after permanent
occlusion of the middle cerebral artery up to 20 h [13]. It may be concluded
that: (1) the hypoxic protocol we used was not sufficiently severe; and/or (2)
other or additional stimuli are required to induce an increase in mRNA. The
HIF-a subunits are structurally similar in their DNA binding and
dimerization domains but different in their transactivation domains, implying
that they may have unique target genes. From studies of two cell lines (HEK293
and 768-O), Hu et al. demonstrated that HIF-2a regulated a variety of
broadly expressed hypoxia-inducible genes and only HIF-1a (not HIF-2a) regulated the
expression of the glycolytic gene during hypoxia [14]. By RNA interference,
Warnecke et al. demonstrated that erythropoietin is a HIF-2a target gene in
Hep3B and Kelly cells [15]. High steady-state staining of HIF-2a mRNA was
detected in the pulmonary artery. Therefore we suggest that HIF-2a plays an
important role in homeostasis in adult rat lung artery. The importance of
HIF-2a versus HIF-1a in hypoxic adaptation needs to be further investigated by
specifically inactivating HIF-1a and HIF-2a.Surprisingly, in contrast to other a-class subunits, a
significant increase in HIF-3a mRNA occurred after 7
d of hypoxia. Unlike HIF-1a and HIF-2a, the moderate decrease in O2 concentration
used in our model was therefore sufficient to induce HIF-3a mRNA synthesis. It was
demonstrated in COS-1 cells that HIF-3a also interacts with HIF-1b, although at
that time, the role of HIF-3a in the cellular response to hypoxia had not been established [16].
There is little evidence that HIF-3a is linked to conditions with low O2
concentration. By transfecting rat hepatocytes with HIF-3a, mRNA
analysis could be shown that HIF-3a mRNA is expressed
predominantly in previous areas of the liver, a fact that was attributed to
the formation of an O2 gradient with lower oxygen concentration
in perivenousareas of the liver caused by the unidirectional flow of blood from
portal to central veins in this organ [17]. Despite the structural similarities
of HIF-3a with other a-class subunits, HIF-3a seems to constitute a more sensitive and
rapidly reacting response component to systemic hypoxia compared with HIF-1a and HIF-2a, as reported
previously [10]. But the exact functional properties of HIF-3a are still
unknown. It can not be excluded that HIF-3a itself may also be
involved in the pathogenesis of hypoxic pulmonary hypertension, by inducing
target genes of HIF-1a, such as HO-1 and VEGF in pulmonary artery [12,13],
especially during early phases of systemic hypoxia. On the other hand, it may
be speculated that HIF-3a acts as a counterpart to other HIF-a subunits. This is
supported by transfection experiments that HIF-3a of human suppresses HIF-a mediated target
gene expression [18]. Therefore, it appears more likely that HIF-3a represents a
subtle and sensitive mechanism that modulates the HIF response even in short
and/or moderate hypoxia. In addition to protein stability, the transactivation
activity of HIF-3a was different from those of HIF-1a and HIF-2a. The
transfection of the HIF-3a protein suppressed HRE-driven gene expression when the expression
of ARNT was limited [7]. The importance of HIF-3a during hypoxia is further
supported by the very recent finding that the HIF-3a gene can be
spliced alternatively in mice [19]. The resulting HIF-3a variant
could be detected only when mice were exposed to severe hypoxia (6% O2),
where it was expressed predominantly in myocardial tissues and lungs. Our
results obtained by Western blot analysis indicate that HIF-2a also undergoes
hypoxic stabilization, which therefore supports a role for it in hypoxia.
Regarding HIF-3a, inconsistent results arose between immunohistochemistry and
Western blot. These controversial results may have risen because of the use of
different sources (pulmonary artery vs. lung homogenate) or because HIF-3a protein
underwent cell-specific stabilization.In conclusion, this study has, for the first time, directly compared
the dynamic expression of HIF-a subunits in pulmonary arteries of rat with hypoxia-induced
hypertension. The findings indicate that additional regulatory steps appear to
operate to determine which alternative subunit is induced. Base on the
differential expression patterns of HIF-1a, HIF-2a and HIF-3a during
prolonged hypoxia, it is tempting to speculate that target gene specificity
plays an important role in hypoxia-induced pulmonary hypertension development.
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