Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 883-892
doi:10.1111/j.1745-7270.2008.00464.x
Expression and role of factor
inhibiting hypoxia-inducible factor-1 in pulmonary arteries of rat with
hypoxia-induced hypertension
Daiyan Fu1,2, Aiguo Dai1*, Ruicheng Hu1, Yunrong Chen1, and Liming Zhu1
1
Department of
Respiratory Medicine, Hunan Institute of Gerontology, Hunan Province Geriatric
Hospital, Changsha 410016, China
2
Graduate School of
University of South China, Hengyang 421001, China
Received: May 22,
2008
Accepted: July 21,
2008
This work was
supported by grants from the National Natural Science Foundation of China (No.
30570815), the Natural Science Foundation of Hunan Province (Nos. 05JJ30073 and
07JJ3035), and the China Postdoctoral Science Foundation (No. 2003033436)
*Corresponding
author: Tel, 86-731-4762793; Fax, 86-731-4735215; E-mail, [email protected]
Hypoxia-inducible
factor-1a subunit (HIF-1a) plays a pivotal role
during the development of hypoxia-induced pulmonary hypertension (HPH) by
transactivating it’s target genes. As an oxygen-sensitive attenuator, factor
inhibiting HIF-1 (FIH) hydroxylates a conserved asparagine residue within the
C-terminal transactivation domain of HIF-1a under normoxia and
moderate hypoxia. FIH protein is downregulated in response to hypoxia, but its
dynamic expression and role during the development of HPH remains unclear. In
this study, an HPH rat model was established. The mean pulmonary arterial
pressure increased significantly after 7 d of hypoxia. The pulmonary artery
remodeling index became evident after 7 d of hypoxia, while the right
ventricular hypertrophy index became significant after 14 d of hypoxia. The
messenger RNA (mRNA) and protein expression of HIF-1a and vascular
endothelial growth factor (VEGF), a well-characterized target gene of
HIF-1a,
were
markedly upregulated after exposure to hypoxia in pulmonary arteries. FIH
protein in lung tissues declined after 7 d of hypoxia and continued to decline
through the duration of hypoxia. FIH mRNA had few changes after exposure
to hypoxia compared with after exposure to normoxia. In hypoxic rats, FIH
protein showed significant negative correlation with VEGF mRNA and VEGF
protein. FIH protein was negatively correlated with mean pulmonary arterial
pressure, pulmonary artery remodeling index and right ventricular hypertrophy
index. Taken together, our results suggest that, in the pulmonary arteries of
rat exposed to moderate hypoxia, a time-dependent decrease in FIH protein may
contribute to the development of rat HPH by enhancing the transactivation of
HIF-1a target genes
such as VEGF.
Keywords hypoxia-inducible factor-1a
subunit; factor inhibiting hypoxia-inducible factor-1; asparaginyl hydroxylase;
hypertension; pulmonary; transactivation
It has been established that chronic hypoxia induces pulmonary
vascular remodeling. Characterized by medial and adventitial thickening due to
increases in cell size and number as well as by increased extracellular matrix
protein accumulation, pulmonary vascular remodeling leads to pulmonary
hypertension. Progressive pulmonary hypertension may result in right
ventricular hypertrophy and eventually in cor pulmonale [1].The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric
transcription factor composed of an a subunit (HIF-1a) and a b subunit (HIF-1b). Because it regulates
the expression of more than 100 genes involved in cellular adaptation and
survival under hypoxia, HIF-1 is one of the most important factors in cellular
response to decreased oxygen availability [2]. Heterozygous HIF-1a deficient
(Hif-1a+/–) mice have impaired pulmonary vascular remodeling after exposure
to 10% O2 for 3 weeks [3]. Our previous studies showed that increased HIF-1a plays a
critical during the development of rat hypoxia-induced pulmonary hypertension
(HPH) by transactivating its target genes, such as inducible nitric oxide
synthase, heme oxygenase-1, vascular endothelial growth factor (VEGF)
and transforming growth factor-b1 [4–7].HIF-1b is a constitutive nuclear subunit, whereas HIF-1a is an
oxygen-sensitive subunit. Under normoxia, hydroxylation of two proline
residues in the oxygen-dependent degradation domain of HIF-1a mediated by
prolyl hydroxylases (PHD) triggers an association with the von Hippel-Lindau
ubiquitin E3 ligase complex, which results in HIF-1a degradation through
ubiquitin-proteasome pathway. However, stabilization of HIF-1a declines in
hypoxia due to PHD inactivation. Subsequently, HIF-1a translocates from the
cytoplasm to the nucleus, where it dimerizes with HIF-1b and binds hypoxia response
elements in its target genes promoters. The binding of HIF-1a and HIF-1b to hypoxia
response elements assists in the recruitment of transcriptional coactivator
p300/CBP, which forms transcription initiation complexes to increase the
transactivation of HIF-1a target genes [8].Factor inhibiting HIF-1 (FIH), also known as asparaginyl
hydroxylase [9], is involved in hydroxylated activity in which a conserved
asparagine residue within the C-terminal transactivation domain (C-TAD) of
HIF-1a suppresses HIF-1a transactivating activity by blocking the binding of the p300/CBP to
HIF-1a C-TAD [10]. In contrast to PHD, FIH can retain its hydroxylated
activity not only in normoxia but also following exposure to moderate hypoxia [11].
It has recently been shown that FIH protein expression decreases in a
time-dependent manner in response to hypoxia [12]. FIH’s dynamic expression
and role as a critical hydroxylase modulating HIF-1a transactivating activity
in HPH have so far not been understood.To evaluate FIH’s expression changes and role in pulmonary arteries
during chronic hypoxia, we investigated the expression patterns of FIH and VEGF,
a well-characterized target gene of HIF-1a [13], as well as their
relationship to each other in the pulmonary arteries of rats at different
phases of HPH development.
Materials and Methods
Materials
The study was approved by the Animal Ethics Committee of the Hunan
Institute of Gerontology (Changsha, China), and abided by Hunan province guidelines
for the care and use of laboratory animals. Hypoxia and normoxia rat models
were set up [14]. Briefly, forty Wistar rats (male, 25020 g, 8–9 weeks old)
purchased from the Animal Experimental Center of Hunan University of
Traditional Chinese Medicine (Changsha, China) were randomly divided into five
groups, with eight rats in each group. Four groups of hypoxic rats were exposed
to normobaric hypoxia at 100.5% O2 for 3, 7, 14 and 21 d (8 h per
day, intermittently), respectively, in a ventilated chamber. 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 MB80
oxygen analyzer (Zhuhai S.E.Z. Hangto Science & Tech. Company, Zhuhai,
China) was used to monitor the chamber’s 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 using soda lime. Excess humidity was prevented using anhydrous calcium
chloride, and ammonia was kept to a minimum level using boric acid in the
chamber. The control rats were kept in a normoxic ventilated chamber (21% O2) in the same room and 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) [4].
mPAP measurement
mPAP was measured [14]. In brief, after rats were
intraperitoneally anesthetized with 40 mg/kg pentobarbital sodium, a specially
designed PE-50 single lumen catheter,(BD Biosciecnces, Sparks, USA) was
inserted into the main pulmonary artery through the right jugular vein. The
injecting position was confirmed by the waveform of pressure. Through this
catheter, mPAP was measured using a Medlab bio-signal operating system
(Nanjing MedEase Science and Technology, Nanjing, China).
Sample preparation and RVHI
measurement
After the measurement of mPAP, the rats were killed and bled out through
their bilateral common carotid arteries. The chest wall was opened, and four
lobes from each right lung were removed. The lobes were used for reverse
transcription-polymerase chain reaction (RT-PCR) and western blot analysis, placed in liquid nitrogen for rapid
freezing and then stored at –80 ?C. The left
lung was used for morphometry, in situ hybridization and immunohistochemical
examination; it was removed and placed in formalin for fixation. Next, the heart was collected for RVHI measurement. For RVHI measurement,
each heart was cut open and the atria were removed. The right ventricular free
wall was dissected, and each chamber was weighed. RVHI was calculated using the
following equation:
Eq.
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.
Morphometric analysis
Sections of 4 mm from each left upper lung were embedded with paraffin, stained
with hematoxylin-eosin and examined under a light microscope. Three tissue sections
were selected from each rat, and at least five representative pulmonary
arteries with an external diameter about 100 mm, also called pulmonary
arterioles, chosen from each section were independently examined. To evaluate
hypoxic remodeling, we calculated the parameters of pulmonary vascular
cross-sections by measuring both the ratio of vascular wall area to total
vascular area (WA) and the ratio of vascular wall thickness to external
diameter (WT). 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, VEGF and FIH mRNA
In situ hybridization was carried out
using an in situ hybridization detection kit (Wuhan Boster Biological
Technology, Wuhan, China). The oligonucleotide probes were designed by Wuhan
Boster Biological Technology according to the sequences of rat HIF-1a, VEGF
and FIH. The sequence of probes against HIF-1a and VEGF
mRNA was the same as that described by Li and Dai [6]. Namely, the sequence of
probes against HIF-1a
mRNA was: 5‘-TTATGAGCTTGCTCATCAGTTGCCACTTCC-3‘;
5‘-CTCAGTTTGAACTAACTGGACACAGTGTGT-3‘; 5‘-GGCCGCTCAATTTATGAATATTATCATGCT-3‘.
And the sequence of probes against VEGF mRNA was: 5‘-GCTCTACCTCCACCATGCCAAGTGGTCCCA-3‘;
5‘-GACCCTGGTGGACATCTTCCAGGAGTACCC-3‘; 5‘-GCAGCTTGAGTTAAACGAACGTACTTGCAG-3‘.
The sequence of probes against FIH mRNA was: 5‘-TTCTCTGTGTACAGTGCCAGCACCCATAAGTTCTT-3‘;
5‘-TTTAACTGGAACTGGATTAATAAACAACAGGGGAA-3‘; and 5‘-CATCAGAAAGTAGCCATCATGAGAAACATTGAGAA-3‘.
Hybridization was carried out on serial tissue sections from the
left lower lung in paraffin fixed by formalin containing 0.1% diethyl
pyrocarbonate (Wuhan Boster Biological Technology) according to the
manufacturer’s instructions. Sections were digested with pepsin for 15 min at
37 ?C. After 2 h pre-hybridization, in which sections were incubated with 20 ml
pre-hybridization solution at 38 ?C in a moist chamber, sections underwent
hybridization with digoxin-labeled, single-stranded oligonucleotide probes for
16 h at 38 ?C. In negative control studies, labeled oligonucleotide probes
were replaced by phosphate-buffered saline. After washing off unbound probes,
sections were incubated first with rabbit antibodies against digoxin and then
with biotinylated goat antibodies against rabbit immunoglobulin G. Sections
were then incubated with streptavidin-biotin-peroxidase. Peroxidase activity
was visualized by color reaction using diaminobenzidine (Wuhan Boster
Biological Technology) as the substrate. Brown and yellow colors indicated
positive results; more specifically, weakly positive and positive staining were
indicated by light yellow and yellow, respectively, and strongly positive is
shown as dark yellow or brown [15]. Negative staining is shown as background
color. Finally, the sections were counterstained with hematoxylin and mounted.
mRNA levels were quantified by the PIPS-2020 pathology image analysis system
based on the absorbance of positive signals from pulmonary arteriole walls.
Three tissue sections were selected from each rat, and at least five
representative pulmonary arterioles chosen from each section were independently
examined.
RT-PCR analysis for FIH
gene
Total RNA from 0.1 g right upper lobe was extracted using TRIzol
reagent (Molecular Research Center, Cincinnati, USA). The concentration of RNA
extracted was determined using a DU-70 UV spectrophotometer (Beckman Coulter,
Fullerton, USA). First-strand complementary DNA (cDNA) was synthesized with
RevertAidTM first-strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania)
according to the manufacturer’s instructions and stored at –20 ?C for
further amplification. The PCR was carried out in a DNA thermal cycler
(Eppendorf, Hamburg, Germany). Reaction mixtures (25 ml) consisted of cDNA (1 ml), 10 mM forward
primer (1 ml), 10 mM reverse primer (1 ml), ddH2O (9.5 ml) and 2?Master Mix (12.5 ml) (Tiangen, Beijing, China). The PCR was performed with the following
thermal profiles: denaturation at 94 ?C for 5 min, followed by 30 cycles of 30
s at 94 ?C, 30 s at 53.5 ?C, 1 min at 72 ?C and extension at 72 ?C for 10 min.
The amplified products were electrophoretically separated with 1.5% agarose
gels. The DNA bands were scanned for optical density values with a densitometer
and quantified using the Tanon gel image system version 3.74 (Shanghai Tanon
Science and Technology, Shanghai, China). The mRNA expression was evaluated
using the ratio of the intensities of the target band to the b-actin band. The
RT-PCR analysis was independently performed in triplicate for each sample. The
primer sequences, as well as the predicted length of the amplified products,
are listed in Table 1. Optimum annealing temperatures are 85 ?C and
numbers of cycles are 30 cycles for amplification.
Immunohistochemistry for HIF-1a, VEGF and FIH protein
A commercial streptavidin-biotin complex kit (Wuhan Boster
Biological Technology) was used for immunohistochemistry [6]. Serial sections
of formalin-fixed, paraffin-embedded left upper lung were digested with 3% H2O2 for 20 min at room temperature, and then preincubated with 10%
non-immunized serum. Sections were incubated with goat anti-HIF-1a, anti-VEGF and
anti-FIH specific polyclonal antibodies (1:100) (Santa Cruz Biotechnology,
Santa Cruz, USA) overnight at 4 ?C. In negative control studies, the antibodies
were substituted by phosphate-buffered saline. After unbound antibodies were
washed off, the sections were incubated with corresponding biotinylated
secondary antibodies against goat and thereafter incubated with streptavidin
peroxidase. Subsequently, peroxidase activity was visualized by a color
reaction with diaminobenzidine. Brown and yellow colors indicated positive
results; more specifically, weakly positive and positive staining were
indicated by light yellow and yellow, respectively, and strongly positive is
shown as dark yellow or brown [15]. Negative staining is shown as background
color. Finally, the sections were counterstained with hematoxylin and mounted.
Protein levels were quantified using the PIPS-2020 pathology image analysis
system based on the absorbance of positive signals in pulmonary arteriole
walls. Three tissue sections were selected from each rat, and at least five
representative pulmonary arterioles chosen from each section were
independently examined.
Western blot analysis for FIH
protein in lung tissue
Proteins from 0.1 g right lower lobes were extracted using a total protein
extraction kit (Nanjing KeyGen Biotech, Nangjing, China) according to the
manufacturer’s instructions. Protein concentrations were determined using the
BCA protein assay kit (Nanjing KeyGen Biotech). Equal amounts of protein were
electrophoresed in 10% sodium dodecylsulfate-polyacrylamide gel, and
transferred to a Hybond enhanced chemiluminescence nitrocellulose membrane
(Amersham, Piscataway, USA). The membranes were blocked with 5% (W/V)
instant non-fat milk in Tris-buffered saline and 0.1% Tween-20 for 1 h at room
temperature and incubated overnight at 4 ?C with the 1:1000 dilution of
specific antibodies of FIH and b-actin (Santa Cruz Biotechnology). Membranes were then incubated
with the 1:2000 dilution of peroxidase-conjugated anti-goat immunoglobulin G
(Wuhan Boster Biological Technology). An enhanced chemiluminescence detection
kit (Santa Cruz Biotechnology) was used for signal detection. Tanon gel image
system version 3.74 was used for quantification. Western blot analysis was
independently performed in triplicate for each sample.
Statistical analysis
Data are expressed as mean±SD. One-way ANOVA was used to determine statistically significant
differences among multiple groups, and the Student-Neuman-Keuls test was used
to analyze statistical significance between two groups. The correlations were
evaluated using the Pearson? product-moment correlation coefficient statistical method. P<0.05 was considered a statistically significant difference.
Results
Chronic hypoxia increases mPAP
mPAP was measured as an indicator of pulmonary artery pressure in
conscious rats. mPAP in normoxic rats was 15.91.3 mmHg. As expected, mPAP in
hypoxic rats increased after 7 d of exposure to hypoxia (P<0.05), reached its peak after 14 d of hypoxia and then remained unchanged (Table 2).
Chronic hypoxia-induced
hypoxic pulmonary vascular remodeling and right ventricular hypertrophy
Pulmonary arterioles in normoxic rats were thin, whereas after
exposure to hypoxia, the wall area and wall thickness of pulmonary arterioles
increased (Table 2). Quantification of these changes, compared with
normoxic controls, revealed that WA increased significantly in hypoxic rats
after 7 d of hypoxia and that the increase of WT became significant after 14 d
of hypoxia. WA and WT increased further with prolonged hypoxia. 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, and it increased further
after 21 d of hypoxia. These data indicated that right ventricular hypertrophy
developed after 14 d of exposure to hypoxia.
mRNA and protein levels of HIF-1a and VEGF in pulmonary
arterial walls during normoxia and chronic hypoxia conditions
In situ hybridization showed that HIF-1a mRNA
expression in pulmonary arteriole walls had significantly increased after 14 d
of hypoxia and remained stable thereafter. However, there were no obvious changes
after 7 d of hypoxia or normoxia (Table 3) (Fig. 1). VEGF
mRNA expression was markedly increased in pulmonary arteriole walls after 7 d
of exposure to hypoxia, reached its peak after 14 d of hypoxia and then
remained stabile (Table 3) (Fig. 2). Expression of HIF-1a and VEGF
mRNA was mainly located in the tunica intima and tunica media.Immunohistochemical analysis showed that HIF-1a protein was
poorly stained in pulmonary arteriole walls of control rats. Yet, staining of
HIF-1a was markedly positive in the tunica intima and tunica media of
pulmonary arterioles after 3 and 7 d of hypoxia, but it then weakened (Table
3) (Fig. 3). In the tunica intima of pulmonary arterioles, VEGF
staining was lightly colored after 3 d of hypoxia and normoxia. However, VEGF
staining became stronger in the tunica intima and tunica media of pulmonary
arterioles after 7 d of hypoxia, increased in strength after 14 d of hypoxia
and then remained stable (Table 3) (Fig. 4).
mRNA and protein levels of FIH
in pulmonary arterial walls during normoxia and chronic hypoxia conditions
The result of in situ hybridization showed that FIH
mRNA was located predominantly in the tunica intima and tunica media of
pulmonary arterioles and changed little after exposure to hypoxia compared with
normoxia (Table 3) (Fig. 5). RT-PCR analysis revealed that FIH
mRNA was clearly expressed in rat lung under normoxic conditions and remained
unchanged in the lungs of all hypoxic rats (Fig. 6).Immunohistochemistry showed that FIH protein was strongly stained in
the tunica intima and tunica media of pulmonary arterioles in normoxic rats.
FIH staining was remarkably weakened in pulmonary arteriole walls, especially
in the tunica media, in rats suffering hypoxic stress for 7 d compared with
controls. Subsequently, this decreased further by 14 d of hypoxia and then
remained stabile (Table 3) (Fig. 7). In western blot analysis, FIH protein levels decreased
significantly after 7 d of hypoxia and lessened further throughout the duration
of hypoxia (Fig. 8).
Analysis of linear correlation
Linear correlation analysis was carried out between different
parameters for hypoxic rats. Linear correlation analysis showed that HIF-1a mRNA, HIF-1a protein, VEGF
mRNA and VEGF protein were positively correlated with mPAP and pulmonary artery
remodeling index (WA and WT). HIF-1a mRNA, VEGF mRNA
and VEGF protein were positively correlated with RVHI. VEGF mRNA and protein
showed significant positive correlations with HIF-1a mRNA and
HIF-1a protein. FIH protein showed significant negative correlations with VEGF
mRNA and VEGF protein. FIH protein was negatively correlated with mPAP,
pulmonary artery remodeling index and RVHI (Table 4).
Discussion
Development of pulmonary hypertension during chronic hypoxia results
from a reduction in vascular caliber because of pulmonary artery remodeling.
The present results demonstrated that hypoxic rats develop pulmonary artery
remodeling after 7 d of exposure to hypoxia, as reflected by the significant
increases in WA, and pulmonary hypertension after 14 d exposure, as revealed
by the rise in mPAP and right ventricular hypertrophy.By enhancing HIF-1a-mediated target genes transactivation, HIF-1a is one of the
pivotal mediators in the pathogenesis of HPH development in rat [4–7]. FIH is an
asparaginyl hydroxylase that regulates the transactivational activity of HIF-1a. In normoxia
and moderate hypoxia, FIH hydroxylates a conserved asparagine residue within
the C-TAD of HIF-1a [11]. This modification blocks binding of transcriptional
coactivator p300/CBP to the HIF-1a C-TAD and inhibits the transactivation of
HIF-1a target genes. FIH is widely expressed and thus potentially
available for the regulation of HIF-1a transactivational activity across a broad
range of cells [11]. As for blood vessels, FIH is mainly expressed in the
smooth muscle cell and endothelium [16]. The present study showed that, when exposed to hypoxia, mRNA and
protein expression of HIF-1a and VEGF increased significantly, and expression of VEGF persisted
for a longer time than that of HIF-1a in pulmonary arteries after the onset of
hypoxia. VEGF mRNA and VEGF protein showed significant positive
correlations with HIF-1a mRNA and HIF-1a protein in hypoxic rats.
These findings were consistent with previous work from our laboratory, which
suggested that HIF-1a may upregulate the expression of VEGF gene by
transactivation, and both HIF-1a and VEGF are involved in the pathogenesis of HPH in rats [6]. The
expression of VEGF, a well-characterized target gene of HIF-1a, has been shown
to be inhibited by FIH [17]. However, in pulmonary arteries of rats suffering
moderate hypoxia (10% O2) in which FIH is active, it is
unclear how does HIF-1a escape the inactivation mechanism.Prior studies have shown that FIH mRNA expression is not
influenced by hypoxia, though its protein levels decline in response to low
oxygen tension [12,18]. In this study, we detected steady-state expression of FIH
mRNA under hypoxia, which was mainly limited to the tunica intima and tunica
media. Nonetheless, over time, FIH protein showed a gradual decrease in hypoxic
rats compared with in controls. This decrease became significant after 7 d of
hypoxia and confirmed results from previous reports [12,18]. Linear correlation
analysis showed that VEGF mRNA and VEGF protein were negatively
correlated with FIH protein. These findings suggest that FIH protein is
downregulated in pulmonary artery under hypoxia, and they also provide a
possible explanation why HIF-1a can transactivate and upregulate the expression of target genes,
such as VEGF, under moderate hypoxia in which FIH is still active.
Moreover, FIH protein was negatively correlated with mPAP, pulmonary artery
remodeling index and RVHI. These results indicate that decreased FIH protein in
pulmonary artery under moderate hypoxia may reduce its inhibitory effect on
HIF-1a transactivational activity and thus on the promotion of
transactivation of HIF-1a target genes, such as VEGF. As a result, FIH is implicated
in the pathogenesis of HPH in rat.In summary, in the pulmonary arteries of rats exposed to moderate
hypoxia, a time-dependent decrease in FIH protein may contribute to development
of rat HPH by enhancing the transactivation of HIF-1a target genes, such as VEGF.
In the present study, FIH protein decreased gradually without significant
changes in the FIH mRNA after the rats were exposed to hypoxia. This
indicates that FIH may be downregulated by some post-transcriptional mechanism.
Fukuba et al recently documented that Siah-1, a member of the E3
ubiquitin ligase family with the really interesting new gene-finger protein
motif, targets FIH for proteasome-dependent degradation under hypoxic
conditions [12]. However, further research is needed to clarify whether Siha-1
contributes to the regulation of FIH during the development of HPH. Therefore,
based on the observations in this study, further elucidation of decreases in
FIH under moderate hypoxia might lead to potential new therapies for the
management of HPH.
References
1 Stenmark KR, Davie NJ,
Reeves JT, Frid MG. Hypoxia, leukocytes, and the pulmonary circulation. J Appl
Physiol 2005, 98: 715721
2 Martinez-Sanchez G,
Giuliani A. Cellular redox status regulates hypoxia inducible factor-1
activity. Role in tumour development. J Exp Clin Cancer Res 2007, 26: 3950
3 Yu AY, Shimoda LA, Iyer
NV, Huso DL, Sun X, McWilliams R, Beaty T et al. Impaired physiological
responses to chronic hypoxia in mice partially deficient for hypoxia-inducible
factor 1alpha. J Clin Invest 1999, 103: 691696
4 Hu R, Dai A, Tan S.
Hypoxia-inducible factor 1 alpha upregulates the expression of inducible nitric
oxide synthase gene in pulmonary arteries of hyposic rat. Chin Med J 2002, 115:
18331837
5 Li QF, Dai AG. Hypoxia
inducible factor-1 alpha correlates the expression of heme oxygenase-1 gene in
pulmonary arteries of rat with hypoxia-induced pulmonary hypertension. Acta
Biochim Biophys Sin 2004, 36: 133140
6 Li QF, Dai AG.
Hypoxia-inducible factor-1 alpha regulates the role of vascular endothelial
growth factor on pulmonary arteries of rats with hypoxia-induced pulmonary
hypertension. Chin Med J 2004, 117: 10231028
7 Jiang Y, Dai A, Li Q, Hu
R. Hypoxia induces transforming growth factor-beta1 gene expression in the
pulmonary artery of rats via hypoxia-inducible factor-1alpha. Acta Biochim
Biophys Sin 2007, 39: 7380
8 Reynolds PR, Mucenski ML,
Le Cras TD, Nichols WC, Whitsett JA. Midkine is regulated by hypoxia and causes
pulmonary vascular remodeling. J Biol Chem 2004, 279: 3712437132
9 Hewitson KS, McNeill LA,
Riordan MV, Tian YM, Bullock AN, Welford RW, Elkins JM et al.
Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor
inhibiting HIF (FIH) and is related to the cupin structural family. J Biol Chem
2002, 277: 2635126355
10 Lando D, Peet DJ, Gorman JJ,
Whelan DA, Whitelaw ML, Bruick RK. FIH-1 is an asparaginyl hydroxylase enzyme
that regulates the transcriptional activity of hypoxia-inducible factor. Genes
Dev 2002, 16: 14661471
11 Stolze IP, Tian YM, Appelhoff
RJ, Turley H, Wykoff CC, Gleadle JM, Ratcliffe PJ. Genetic analysis of the role
of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (HIF)
in regulating HIF transcriptional target genes. J Biol Chem 2004, 279:
4271942725
12 Fukuba H, Yamashita H, Nagano
Y, Jin HG, Hiji M, Ohtsuki T, Takahashi T et al. Siah-1 facilitates
ubiquitination and degradation of factor inhibiting HIF-1alpha (FIH). Biochem
Biophys Res Commun 2007, 353: 324329
13 Carbia-Nagashima A, Gerez J,
Perez-Castro C, Paez-Pereda M, Silberstein S, Stalla GK, Holsboer F et al.
RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes
HIF-1alpha during hypoxia. Cell 2007, 131: 309323
14 Chen YR, Dai AG, Hu RC, Jiang
YL. Differential and reciprocal regulation between hypoxia-inducible
factor-alpha subunits and their prolyl hydroxylases in pulmonary arteries of
rat with hypoxia-induced hypertension. Acta Biochim Biophys Sin 2006, 38:
423434
15 Hu RC, Dai AG, Tan SX. Study of
the expression of hypoxia-inducible factor 1alpha in lung tissues of patients
with chronic obstructive pulmonary disease. Zhonghua Jie He He Hu Xi Za Zhi
2002, 25: 491492
16 Soilleux EJ, Turley H, Tian YM,
Pugh CW, Gatter KC, Harris AL. Use of novel monoclonal antibodies to determine
the expression and distribution of the hypoxia regulatory factors PHD-1, PHD-2,
PHD-3 and FIH in normal and neoplastic human tissues. Histopathology 2005, 47:
602610
17 Dayan F, Roux D, Brahimi-Horn
MC, Pouyssegur J, Mazure NM. The oxygen sensor factor-inhibiting
hypoxia-inducible factor-1 controls expression of distinct genes through the
bifunctional transcriptional character of hypoxia-inducible factor-1alpha.
Cancer Res 2006, 66: 36883698
18 Metzen E, Berchner-Pfannschmidt
U, Stengel P, Marxsen JH, Stolze I, Klinger M, Huang WQ et al.
Intracellular localisation of human HIF-1 alpha hydroxylases: implications for
oxygen sensing. J Cell Sci 2003, 116: 1319–1326