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-Estradiol decreases hypoxic induction of erythropoietin
gene expression
Vascular Physiology Group, Department of Cell Biology and Physiology, Health Sciences Center, University of New Mexico, Albuquerque, New Mexico 87131-5218
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Exposure to chronic hypoxia induces
erythropoietin (EPO) production to facilitate oxygen delivery to
hypoxic tissues. Previous studies from our laboratory found that
ovariectomy (OVX) exacerbates the polycythemic response to hypoxia and
treatment with 17
-estradiol (E2-) inhibits this effect. We
hypothesized that E2- decreases EPO gene expression during hypoxia.
Because E2- can induce nitric oxide (NO) production and NO can
attenuate EPO synthesis, we further hypothesized that E2- inhibition
of EPO gene expression is mediated by NO. These hypotheses were tested
in OVX catheterized rats treated with E2- (20 µg/day) or vehicle
for 14 days and exposed to 8 or 12 h of hypoxia (12%
O2) or normoxia. We found that E2- treatment significantly decreased EPO synthesis and gene expression during hypoxia. E2- treatment did not induce endothelial NO synthase (eNOS)
expression in the kidney but potentiated hypoxia-induced increases in
plasma nitrates. We conclude that E2- decreases hypoxic induction of
EPO. However, this effect does not appear to be related to changes in
renal eNOS expression.
nitric oxide; polycythemia; hypoxia
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CHRONIC EXPOSURE TO HYPOXIA can cause a number of pathophysiological conditions including pulmonary hypertension and chronic mountain sickness. Hypoxic induction of erythropoiesis leads to polycythemia, which may contribute to the development of these diseases. Erythropoiesis is mediated by the small glycoprotein hormone erythropoietin (EPO; Refs. 3, 12). In adults, EPO is primarily synthesized in the kidney and stimulates red blood cell synthesis in bone marrow. During hypoxia, elevated red blood cell numbers help to compensate for compromised oxygen delivery to tissues.
The physiological and biochemical properties of EPO have been
extensively studied, but there are few studies to investigate gender
differences in regulation of the hormone. It has been demonstrated that
17-estradiol (E2-) can influence the expression of
hypoxia-inducible genes such as vascular endothelial growth factor and
endothelin-1 (ET-1) (4, 11, 58). However, estrogen
regulation of EPO gene expression is not well understood. In some
tissues such as the uterus and ovaries, E2- has been shown to induce
EPO production (34, 67). However, this regulation seems to
be independent of pathways involved in hypoxic stimulation of EPO
synthesis. In contrast, several investigators have shown that women
living at high altitude have a lower hematocrit compared with men at the same altitude. Peschle et al. (40) observed that
administration of estrogen benzoate (10 µg/day) to ovariectomized
(OVX) rats attenuated increases in plasma iron following hypoxia.
Studies from our laboratory (46) have also shown that OVX
augments hypoxia-induced increases in hematocrit in rats (0.5 atm, 4 wk) and that E2- prevents this augmentation. These findings are
consistent with the hypothesis that E2- decreases hypoxic induction
of EPO gene expression.
One potential mechanism for E2- regulation of EPO is through
activation of nitric oxide (NO) synthesis. E2- has been shown to
induce NO production by both genomic and nongenomic mechanisms (1, 27). In addition, NO can reduce EPO gene induction
during hypoxia (62). Therefore, we hypothesize that E2-
decreases EPO gene expression by increasing NO synthesis in OVX rats.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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All animal protocols were reviewed and approved by the University of New Mexico Institutional Animal Care and Use Committee.
Animal preparation.
Female Sprague-Dawley rats (body wt 250-300 g; Harlan Industries)
were anesthetized with a mixture of ketamine (100 mg/kg im) and
acepromazine (1 mg/kg im). Ovaries were removed via bilateral laparotomy. Rats were allowed 2 wk for recovery and depletion of
endogenous estrogen stores. After recovery, femoral arterial and venous
catheters were surgically implanted, and mini osmotic pumps (Alzet
model 2002) containing lipid-soluble E2- (20 µg/24 h) or vehicle
(polypropylene glycol) were implanted subcutaneously at the base of the
neck (46). We have previously shown that this dose of
E2- yields plasma levels of the hormone of 102 ± 11 pg/ml in
the E2--treated animals compared with <5 pg/ml in the
vehicle-treated animals (17, 18). These levels are within the physiological range of circulating estrogen in the rat (5, 7,
30). One week after catheter surgery, rats were placed in a
Plexiglas chamber (25 × 15 × 10 cm) that contained fresh bedding and were allowed to adapt to their new environment for 30 min
before experimentation. After this adjustment period, the chamber was
flushed with room air or a hypoxic gas (12% O2) for 8 or
12 h. The percent oxygen in the chamber was monitored continually with an oxygen analyzer (model S-3A/1, Applied Electrochemicals). Plasma samples were collected at 0, 4, 8, and 12 h of exposure. Only animals with hematocrit 
38% postcatheterization were used, because a decrease in hematocrit could induce EPO production. To avoid
decreases in hematocrit during the experimental period, red blood cells
were resuspended in saline and infused back into the animal after each
sample collection. Uterine weight was measured as an indicator of
E2- delivery (14, 31, 39). At the final time point,
blood gases were measured to confirm exposure conditions using a blood
gas analyzer (model ABL-5, Radiometer).
Radioimmunoassay.
Plasma samples were collected through the arterial catheter at 0, 4, 8, and 12 h. EPO protein was assayed using a double-antibody RIA kit
(DiaSorin). This kit is a competitive-binding, disequilibrium RIA
utilizing recombinant human EPO for both tracer and standards and a
goat anti-EPO primary antibody (EPO-trac). Human and rat EPO cDNA share
>80% homology including some very highly conserved regions
(66). This RIA, although based on human EPO, has been used
for analysis of EPO from other species (26, 33) including rat plasma (22-24), and the antibody is 100%
cross-reactive with rat EPO. The manufacturer (DiaSorin) reports a
percent covariance of within-assay variability across 20 replicates as
9.8 for samples ranging in concentration from 11.1 to 254.6 mU/ml. We
conducted assays in triplicate to minimize within-assay variability.
For the same range of sample concentrations, the between-assay
variability is reported by the manufacturer as percent covariance of
11.88. The between-assay variability for four separate assays (data in Fig. 1) was 21.68%. In addition, a
plasma sample from a hypoxic rat was used as an internal control for
interassay variability and to normalize data by dividing all samples by
this value for each assay. The differences between groups were
statistically significant with or without normalization. The minimum
detectable concentration of EPO was 4.4 mU/ml and the maximum detection
limit was 300 mU/ml. Linearity with dilution is reported by the
manufacturer for samples from 2- to 100-fold dilution. We evaluated
linearity in three rat plasma samples. Linearity was maintained with
10-fold dilution in these samples.
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RNA isolation. Total RNA was extracted from the kidneys of rats treated as outlined using the TRIzol extraction method described elsewhere (36). Briefly, kidneys were homogenized, total RNA was extracted and precipitated with isopropyl alcohol, washed in ethanol, and resuspended in RNase- and DNase-free water. RNA quantity and quality were determined by spectrophotometry and agarose gel electrophoresis, respectively.
Real-time quantitative PCR. EPO mRNA levels were determined by one-step real-time RT-PCR. Probes and primer sequences were generated from the rat EPO sequence using Primer Express software. Total RNA was quantified in real time using a double dye-labeled fluorogenic oligonucleotide probe (53) and an automated fluorescence-based detection system. The probe was labeled 5' with a fluorogenic reporter dye, 6-carboxy-fluoresceine (FAM), and 3' with a quencher dye, 6-carboxy-tetramethylrhodamine (TAMRA, generated by PE Biosystems). The nucleotide sequence of the probe 5'-(FAM)-AAGCCGCTCCACTCCGAACACTCA-(TAMRA)-3' corresponds to segment 471-494 of rat EPO mRNA. The primer sequences specific for rat EPO cDNA are: forward primer: 5'-AAGCCATCAGTGGGCTACGT-3'; and reverse primer: 5'-CCGGAAGAGCTTGCAGAAAGTA-3'. One-step RT-PCR was performed in 96-well optical plates and run on a TaqMan (ABI Prism 7700 Sequence Detection System, PE Applied Biosystems). Reverse transcription was carried out at 48°C for 30 min followed by a 40-cycle PCR. The PCR cycle used was 95°C for 10 min, 95°C for 15 s, and 60°C for 1 min. The reaction mixture contained magnesium chloride (5.5 mM), forward and reverse primers (10 µM each), probe (2 µM), dATP, dCTP, dGTP, and dTTP (10 µM each), RNase inhibitor (20 U/µl), AmpliTaq Gold DNA polymerase (5 U/µl), and mULV reverse transcriptase (50 U/µl). Data were analyzed using the sequence-detection system software program provided with the TaqMan. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was measured as an internal control using a rodent primer and probe kit. Although it has been previously shown that GAPDH mRNA is elevated upon exposure to hypoxia (69), we did not observe any change in expression under any of our experimental conditions. The GAPDH kit uses a different dye (Vic) as the fluorescent signal and TAMRA as the quencher. There was no difference in the amplification of GAPDH mRNA between experimental groups. All reagents were from PE Applied Biosystems unless otherwise stated.
Determination of plasma levels of NO by chemiluminescence detection. Plasma nitrates and nitrites (NOx) were measured in samples collected at 0, 4, 8, and 12 h using a Sievers 270B NO chemiluminescence analyzer (65). Plasma was diluted in a 1:10 ratio in PBS containing 5% dextran. Dilute sample (100 µl) was injected into the reaction chamber containing a solution of vanadium chloride in concentrated HCl at 90°C. The chamber also contained an antifoaming agent to minimize protein-induced frothing. NOx in the sample was reduced under these conditions and the NO generated was translated to voltage by a photomultiplier tube. Plasma NOx concentrations were calculated from a sodium nitrate standard curve. Final data are expressed as micromoles NOx.
Determination of renal NO synthase expression by Western
blotting.
Expression of all three NO synthase (NOS) isoforms was evaluated in
kidneys using Western blots (46). Frozen tissue was homogenized in ice-cold homogenization buffer [Tris · HCl
buffer containing EDTA (0.3 mg/ml), leupeptin (5 µg/ml), pepstatin A (0.7 µg/ml), aprotinin (2 µg/ml), and phenylmethylsulfonyl fluoride (20 µg/ml)]. Homogenates were spun for 10 min at 1,000 g
at 4°C, and the supernatant was assayed for protein concentration
with the Bradford method (Bio-Rad protein assay) and then separated in
15% polyacrylamide gels. Separated proteins were blocked and probed
with a monoclonal antibody specific for one of the three NOS isoforms
(1:1,000 dilution, Transduction Laboratories). In a parallel set of
experiments, the proteins separated on the gel were probed with an
antibody for 
-actin (1:5,000 dilution, Transduction Laboratories).
Enhanced chemiluminescence was used to visualize labeled protein and
the relative quantity of protein was determined with SigmaGel software.
Densitometric units were quantitated by normalization to the standard
run on the same gel and to the density of -actin bands. Molecular
weight markers and respective protein standards were used to confirm
specific NOS isoform detection and to normalize between gels. In a
separate experiment, 5, 10, 20, 30, 40, and 50 µg of protein/lane
were analyzed to ensure linearity.
Fixation of kidneys for immunohistochemical analysis.
Rat kidneys were fixed for immunohistochemistry by the paraformaldehyde
perfusion technique described elsewhere (45). Briefly, rats were anesthetized with pentobarbital sodium (15 mg/kg iv), the
chest was opened, and 0.1 ml of heparin was injected into the left
ventricle. The ascending aorta was cannulated and the systemic
circulation was flushed for 10 min at a flow rate of 48 ml/min with
perfusion buffer [physiological saline solution containing bovine
serum albumin (4%), papaverine (10
4 M), and heparin (0.3 ml/kg body wt)] and then for 10-25 min with a fixative solution
[PBS containing paraformaldehyde (4%), glutaraldehyde (0.1%), and
papaverine (104 M)]. Cross sections of kidney (4-5
mm) were immersed in fixative solution for 4 h at room
temperature. After fixation, slices were cryoprotected overnight in PBS
containing 20% sucrose before immunostaining.
Immunostaining for renal endothelial NOS and EPO.
Renal EPO and endothelial NOS (eNOS) protein expression were further
analyzed by immunohistochemistry (18, 21). Rat kidney slices were placed in specimen molds containing embedding medium (Tissue-Tek OCT compound) and frozen in isobutane cooled with liquid
nitrogen. Transverse sections (10 µm) were cut and thaw-mounted on
glass (Superfrost Plus) slides. Sections were treated with 0.33%
hydrogen peroxide to inhibit endogenous peroxidases, blocked with
buffer containing both goat and horse serum (4% each), and incubated
with a combination of a mouse monoclonal antibody for eNOS (1:1,000
dilution, Transduction Laboratories) and a rabbit polyclonal antibody
for EPO (1:500 dilution, Santa Cruz Biotechnologies) for 1 h at
room temperature followed by overnight incubation at 4°C. Sections
were subsequently probed with a mixture of a rat-adsorbed, biotinylated
horse anti-mouse IgG (1:400 dilution, Vector Laboratories) and
human-adsorbed alkaline phosphatase-labeled goat anti-rabbit IgG (1:400
dilution, Stressgen) for 1 h at room temperature. Antibody-labeled proteins were stained using a Vector Blue staining kit (Vector Laboratories) for EPO followed by incubation with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride dihydrate (0.07%) in hydrogen peroxide (0.002%) for eNOS. Sections were dehydrated, cleared with a xylene-free clearing medium (Fisher Laboratories), and
permanently mounted using Vectamount xylene-free mounting medium
(Vector Laboratories). Nonspecific binding was evaluated by
substituting total mouse IgG and rabbit serum for the primary antibodies. Densitometric analysis of immunocytochemical labeling was
performed as described previously (44). Staining intensity is expressed in optical density (OD) units according to the calculation
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Data analysis and statistics.
Data are reported as means ± SE. Two- or three-way ANOVA with
Tukey's post hoc test were used where applicable to compare between
groups and treatments. Values for n represent numbers of
animals. A P value of 
0.05 was considered
statistically significant.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Arterial blood gases, hematocrit, and uterine weight.
Table 1 illustrates arterial blood gases
after 8 h of hypoxic or normoxic exposure. Exposure to hypoxia
caused a similar decrease in arterial PO2 in
both vehicle- and E2--treated groups. In addition, arterial
PCO2 decreased and pH increased as anticipated with hypoxic stimulation of ventilation. E2- has been shown to be a
ventilatory stimulant (2). However, there were no
significant differences in blood gas values between the two groups
under either normoxic or hypoxic conditions, which suggests that
ventilatory stimulation by E2- did not mediate the observed effect
on EPO synthesis.
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or vehicle
treatment. Average uterine weight was significantly greater in
E2--treated animals than in control animals (E2-, 0.562 ± 0.011 vs. controls, 0.097 ± 0.005 g; n = 12 animals). Increased uterine weight has been previously used as an
indicator of E2- treatment (14, 31, 39).
Plasma EPO.
Exposure to hypoxia caused a significant increase in plasma EPO in both
the E2--treated and vehicle-treated groups (Fig. 1). A significant
increase was seen after 4 h of hypoxia with a further increase
after 8 h of hypoxia in the vehicle-treated group. However, there
was no further increase in the E2--treated group at 8 h
compared with 4 h, and plasma levels of EPO were significantly
lower in the E2--treated compared with vehicle-treated rats at this
time point (Fig. 1).
persistently decreases or only delays the
onset of EPO synthesis, animals were exposed to 12 h of hypoxia
and plasma EPO levels measured by RIA. We found that exposure to
12 h of hypoxia did not further increase plasma EPO in either group. Rather, the difference in plasma levels between the
E2--treated and vehicle-treated groups was maintained (Fig. 1).
There were no changes in plasma EPO levels during normoxia.
Renal EPO mRNA.
To determine whether E2- attenuation of EPO synthesis was mediated
at the level of gene expression, EPO mRNA levels were determined in
kidneys from animals treated with E2- or vehicle and exposed to
hypoxia or normoxia. GAPDH mRNA was used as an internal control. It has
been previously reported that GAPDH mRNA is induced upon hypoxic
exposure and estrogen treatment (47, 48). However, we did
not observe any difference in gene expression under any of our
experimental conditions. Real-time PCR analysis revealed that exposure
to 8 h of hypoxia significantly increased EPO mRNA in both the
E2-- and vehicle-treated groups (Fig.
2). However, the increase was
significantly greater in vehicle-treated animals compared with those
treated with E2-. Renal EPO mRNA levels were lower after 12 h
of hypoxia compared with 8 h of hypoxia, although they were still
significantly elevated compared with normoxic groups. These results are
consistent with plasma EPO data and previous observations in the
literature (29, 54).
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Plasma NOx.
Treatment with E2- can induce NOS gene expression in a number of
organs including the kidney (1, 18). Also, E2- can directly stimulate NOS activity, thereby increasing NO availability (27). To examine whether E2- treatment increases NO
synthesis, plasma levels of NOx were determined by chemiluminescence
detection. Interestingly, we found that hypoxia elevated plasma NOx in
both E2-- and vehicle-treated groups exposed to hypoxia (Fig.
3). This hypoxic increase was further
augmented by E2- treatment, although no significant effect of E2-
was observed in normoxic rats. Exposure of animals to 12 h of
hypoxia did not cause any further increase in plasma NOx in either
group. However, plasma NOx remained elevated in both groups compared
with the normoxic counterparts. These data suggest that E2-
treatment increases NO release but do not evaluate the source of the
elevated NO production. Therefore, Western blotting was employed to
determine whether E2- upregulates expression of renal NOS isoforms.
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Western blotting for renal NOS isoforms.
To further investigate the hypothesis that E2--induced NO production
mediates the attenuation of EPO induction, expression levels of all
three NOS isoforms in the kidney were analyzed by Western blotting.
However, we did not detect either the inducible or neuronal isoforms of
NOS (iNOS and nNOS, respectively), although we did detect the
respective positive controls (rat brain homogenates for nNOS and
macrophage lysate for iNOS, Transduction Laboratories; lung homogenate
from a lipopolysaccharide-treated rat was used as a second positive
control for iNOS). In all kidney homogenates, eNOS was
detected. However, we did not observe any difference in protein
expression between groups (Fig.
4A). This was further evaluated by immunohistochemistry.
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Immunohistochemistry for renal eNOS and EPO.
E2- regulation of eNOS expression in the kidney was further
evaluated by immunohistochemistry. The double-staining technique also
evaluated the relative proximity of EPO- and eNOS-expressing cells in
the kidney. Kidney eNOS was detected by staining with 3,3'-diaminobenzidine tetrahydrochloride dihydrate with peroxide, whereas EPO was detected with Vector-blue alkaline phosphatase staining
(Fig. 5). Specific staining for
eNOS was observed primarily in endothelial cells of renal arteries.
However, consistent with the Western analyses, there was no induction
of eNOS expression with E2- treatment (Fig. 4B). On the
contrary, eNOS expression tended to be lower in both normoxic and
hypoxic E2--treated groups compared with the respective vehicle
controls, although this difference was only significant between
normoxic groups.
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-treated animals compared with
vehicle-treated rats after hypoxia (Fig. 5B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Previous studies from our laboratory and others led us to
hypothesize that E2- decreases hypoxic induction of EPO in rats by
increasing NO production. The major findings from this study are
1) E2- decreased hypoxic induction of plasma EPO and
renal EPO mRNA expression in OVX rats (see Figs. 1 and 2);
2) exposure to hypoxia increased plasma NOx, and this
induction was further augmented by E2- treatment (see Fig. 3); and
3) E2- did not increase immunoreactive renal eNOS (see
Fig. 4A).
Immunohistochemistry studies confirm that hypoxic induction of EPO was
diminished in tissues from rats treated with E2- and that there was
no increase in renal eNOS protein expression with either E2-
treatment or hypoxia (Figs. 4B and 5).
Reports on estrogen regulation of EPO synthesis during hypoxia are
controversial. As early as 1941, Vollmer and Gorden (referenced in 20)
observed that OVX increases the rate of erythropoiesis in rats. More
recently, it was demonstrated that hypoxia-induced increases in
hematocrit are greater in male Hilltop Sprague-Dawley rats compared
with their female counterparts (38). Moore et al.
(35) have shown a decreased polycythemic response to
hypoxia in female rats compared with males. We have also observed that OVX exacerbates hypoxia-induced increases in hematocrit and that E2-
treatment inhibits this effect of OVX (46). In contrast, others have demonstrated (41) that estrogen does not
affect or actually enhances the polycythemic response to hypoxia. The purpose of this study was to determine whether estrogen regulates hypoxic induction of red blood cell synthesis as evidenced by changes
in EPO synthesis. These data indicate that E2- inhibits EPO
induction during hypoxia by decreasing gene expression.
There are several possible mechanisms by which E2- may inhibit EPO
gene expression during hypoxia. One is through increased NO synthesis.
It has been previously shown that all three NOS isoforms are expressed
in the kidney (63) and that E2- can induce NOS gene
expression in the kidney (42, 43) and other organ systems
(32, 56). Furthermore, E2- has been shown to acutely
increase eNOS activity that results in elevated production of NO
(1, 27). However, others have shown (49, 64)
that E2- treatment does not affect expression of renal NOS isoforms. NO regulation of EPO synthesis is also much debated. NO has been reported to attenuate EPO gene expression under multiple conditions (20, 57), although some investigators have demonstrated
that NO can induce EPO synthesis during hypoxia in Hep3B cells
(37, 68). We therefore hypothesized that E2- increases
NO production to attenuate EPO gene expression. However, Western
analyses and immunohistochemistry studies demonstrate a tendency for
renal eNOS protein expression to be decreased, not increased, with
E2- treatment. Given that NO induces EPO production during hypoxia (37), it is possible, although speculative, that decreased
eNOS expression during E2- treatment contributes to decreased EPO synthesis during hypoxia. In any case, these results clearly indicate that E2--mediated attenuation of EPO synthesis does not require increased renal eNOS protein expression.
Hypoxic induction of EPO gene expression is mediated by the
transcription factor hypoxia inducible factor-1 (HIF-1). Although several investigators have shown that NO attenuates HIF-1
activity/stability during hypoxia thereby decreasing expression of
hypoxia-induced genes (57), other studies suggest that
HIF-1 is stabilized in the presence of NO (28, 50,
51). Therefore, it is possible that the apparent
decrease in renal eNOS protein expression contributed to downregulation
of HIF-1-dependent EPO synthesis in the estrogen-treated rats.
Estrogen has also been shown to stimulate eNOS activity by nongenomic
mechanisms (21). Indeed, in the current study, plasma NOx
was increased after hypoxia, and this effect was augmented by E2-
treatment. Therefore, it appears that E2- augments hypoxia-induced NO production and this augmentation correlates with the decrement in
EPO synthesis. However, it is not clear whether E2--induced NOx is
responsible for attenuation of EPO gene expression during hypoxia.
The present study examined E2--induced NO as one potential mechanism
by which E2- may decrease EPO gene expression during hypoxia.
However, there are other mechanisms by which this might occur. A recent
study from our laboratory demonstrates that E2- reduces
HIF-1-mediated ET-1 gene expression (11). Because hypoxic regulation of both ET-1 and EPO is mediated by HIF-1, it is possible that E2- regulates both genes directly through interaction with the
common factor, HIF-1. Alternatively, E2- may interfere with EPO gene
expression by directly binding to the steroid hormone response element
present in the EPO promoter (25).
Previous studies have shown that E2- and other ovarian hormones
function as ventilatory stimulants. It has been observed that alveolar
ventilation and hypoxic ventilatory responses are greater in females
than in males and such differences have been attributed to ovarian
hormones (2). It has also been speculated that
physiological levels of female hormones act peripherally to raise
carotid body chemosensitivity (60), although the combined administration of estrogen and progestin is a more effective stimulus for increased ventilation (15, 59). To evaluate whether
E2- administration stimulated ventilatory responses in our
experiments, arterial PO2,
PCO2, and pH were measured in vehicle- and
estrogen-treated animals at the end of each experiment (see Table 1).
We did not observe any apparent ventilatory stimulation by estrogen,
and thus a decrease in the degree of hypoxemia could not account for the reduced EPO synthesis in the E2--treated group.
Recent studies by Masuda and others (10, 34) have shown
that EPO is synthesized in a number of organs outside the liver and
kidney. Furthermore, EPO synthesis is under tonic regulation by E2-
in some of these organs (34, 52, 53). In the uterus and
ovaries, E2- is required for hypoxic induction of EPO, which promotes angiogenesis (8). However, in the same study,
E2- did not affect hypoxic induction of EPO in the murine kidney. Although the results from this study are in contrast to ours, it must
be noted that the experimental conditions in the two studies were very
different. The mouse study examined the effect of acute estrogen
treatment on renal EPO synthesis during hypoxia. The mice were given a
bolus of estrogen 30 min before the hypoxic exposure, and the
experiment was designed to compare estrogen induction of EPO in the
kidney with that in the ovaries. Therefore, it is difficult to tell
whether chronic estrogen treatment affects EPO mRNA synthesis in mice
because that was not reported (53). These studies do
suggest that more than one pathway regulates EPO production and that
EPO, in turn, may regulate multiple physiological conditions.
Furthermore, E2- may regulate EPO synthesis in an organ-dependent
and/or a species-dependent manner.
In the present study, a whole-animal model was used to examine the
effect of E2- on EPO gene expression. The results were verified by
several techniques, one of which was immunohistochemical analysis. EPO
protein was immunolabeled in proximal tubule cells of the rat kidney.
However, the site of EPO synthesis within the kidney is much debated.
Earlier studies have suggested multiple sites of EPO synthesis
including the glomerulus and distal tubules. More recently, several
investigators demonstrated EPO mRNA localized only in peritubular
capillary beds of hypoxic kidneys (9, 13). Still others
have demonstrated EPO synthesis in interstitial cells of peritubular
capillaries (16). In our study, staining clearly demonstrated specific immunoreactivity only in proximal tubule cells of
rat kidneys as confirmed by hematoxylin-eosin staining in parallel
sections. There was no EPO staining in serum controls and little
staining in normoxic sections, which suggests that the staining is
indeed EPO specific. However, it is possible that the protein staining
is an artifact of filtration rather than EPO synthesis, and several
investigators have considered this point in evaluating EPO synthesis
within the kidney. However, similar to our studies, both EPO protein
and mRNA have been localized in renal proximal tubule cells (19,
55). Therefore, the probability that this is the site of EPO
synthesis cannot be excluded either. In conclusion, the exact site(s)
of EPO synthesis in the kidney is not clear, although it is interesting
to note that several other secretory products including angiotensinogen
and vitamin D3 are synthesized in proximal tubule cells
(6), which indicates that these cells can act as sites of
hormone synthesis (61).
In conclusion, in vivo hypoxic induction of EPO is decreased by E2-.
This finding is of significance because EPO induction and consequent
polycythemia may contribute to the pathology of diseases like chronic
mountain sickness and pulmonary hypertension. Therefore, estrogen
attenuation of EPO induction may underlie some gender differences in
the development of pulmonary diseases, and estrogen status might be
important in the systemic response to hypoxia.
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ACKNOWLEDGEMENTS |
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The authors thank Pam Allgood and Anna Holmes for technical assistance, Michelle Martinez for help with real-time PCR studies, and Tamara Howard and Dr. Alexis Harris for assistance with immunohistochemistry studies. The authors also thank Dr. Leif Nelin for help with the NOx and chemiluminescence studies and Dr. Theresa O' Donaughy for help with many aspects of the studies.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-03852 (to N. L. Kanagy) and RR-164808 (to T. C. Resta), American Heart Association Predoctoral Fellowship 0110279Z (to H. Mukundan), and by a Scientist Development Grant from the American Heart Association (to T. C. Resta). T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.
Address for reprint requests and other correspondence: H. Mukundan, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico, Health Sciences Center, 915 Camino de Salud, NE, Albuquerque, NM 87131-5218 (E-mail: hmukundan{at}salud.unm.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 4, 2002;10.1152/ajpregu.00573.2001
Received 19 September 2001; accepted in final form 28 March 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Adhikary, G,
Premkumar DR,
and
Prabhakar NR.
Dual influence of nitric oxide on gene regulation during hypoxia.
Adv Exp Med Biol
475:
285-292,
2000.
2.
Aitken, ML,
Franklin JL,
Pierson DJ,
and
Schoene RB.
Influence of body size and gender on control of ventilation.
J Appl Physiol
60:
1894-1899,
1986.
3.
Bauer, C.
The oxygen sensor that controls EPO production: facts and fancies.
J Perinat Med
23:
7-12,
1995.
4.
Bausero, P,
Ben Mahdi M,
Mazucatelli J,
Bloy C,
and
Perrot-Applanat M.
Vascular endothelial growth factor is modulated in vascular muscle cells by estradiol, tamoxifen, and hypoxia.
Am J Physiol Heart Circ Physiol
279:
H2033-H2042,
2000.
5.
Butcher, RL,
Collins WE,
and
Fugo NW.
Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17 throughout the 4-day estrous cycle of the rat.
Endocrinology
94:
1704-1708,
1974.
6.
Chan, JS,
Wang TT,
Zhang SL,
Chen X,
and
Carriere S.
Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.
Mol Cell Biochem
212:
73-79,
2000.
7.
Cheng, DY,
Feng CJ,
Kadowitz PJ,
and
Gruetter CA.
Effects of 17-estradiol on endothelium-dependent relaxation induced by acetylcholine in female rat aorta.
Life Sci
55:
L187-L191,
1994.
8.
Chikuma, M,
Masuda S,
Kobayashi T,
Nagao M,
and
Sasaki R.
Tissue-specific regulation of erythropoietin production in the murine kidney, brain, and uterus.
Am J Physiol Endocrinol Metab
279:
E1242-E1248,
2000.
9.
Da Silva, JL,
Schwartzman ML,
Goodman A,
Levere RD,
and
Abraham NG.
Localization of erythropoietin mRNA in the rat kidney by polymerase chain reaction.
J Cell Biochem
54:
239-246,
1994.
10.
Digicaylioglu, M,
Bichet S,
Marti HH,
Wenger RH,
Rivas LA,
Bauer C,
and
Gassmann M.
Localization of specific erythropoietin binding sites in defined areas of the mouse brain.
Proc Natl Acad Sci USA
92:
3717-3720,
1995.
11.
Earley, S,
and
Resta TC.
Estradiol attenuates hypoxia-induced endothelin-1 gene expression.
Am J Physiol Lung Cell Mol Physiol
283:
L86-L93,
2002.
12.
Eckardt, KU.
The ontogeny of the biological role and production of erythropoietin.
J Perinat Med
23:
19-29,
1995.
13.
Eckardt, KU,
Koury ST,
Tan CC,
Schuster SJ,
Kaissling B,
Ratcliffe PJ,
and
Kurtz A.
Distribution of erythropoietin producing cells in rat kidneys during hypoxic hypoxia.
Kidney Int
43:
815-823,
1993.
14.
Evans, MJ,
Eckert A,
Lai K,
Adelman SJ,
and
Harnish D.
Reciprocal antagonism between estrogen receptor and NF-
B activity in vivo.
Circ Res
89:
823-830,
2001.
15.
Favier, R,
Spielvogel H,
Caceres E,
Rodriguez A,
Sempore B,
Pequignot J,
and
Pequignot JM.
Differential effects of ventilatory stimulation by sex hormones and almitrine on hypoxic erythrocytosis.
Pflügers Arch
434:
97-103,
1997.
16.
Fisher, JW,
Koury S,
Ducey T,
and
Mendel S.
Erythropoietin production by interstitial cells of hypoxic monkey kidneys.
Br J Haematol
95:
27-32,
1996.
17.
Gonzales, RJ,
and
Kanagy NL.
Endothelium-independent relaxation of vascular smooth muscle by 17-estradiol.
J Cardiovasc Pharmacol
4:
227-234,
1999.
18.
Gonzales, RJ,
Walker BR,
and
Kanagy NL.
17-Estradiol increases nitric oxide-dependent dilation in rat pulmonary arteries and thoracic aorta.
Am J Physiol Lung Cell Mol Physiol
280:
L555-L564,
2001.
19.
Haidar, MA,
Loya F,
Yang Y,
Lin H,
Glassman A,
Goldwasser E,
and
Albitar M.
Electron microscopic localization of lacZ expression in the proximal convoluted tubular cells of the kidney in transgenic mice carrying chimeric erythropoietin/lacZ gene constructs.
J Struct Biol
118:
220-225,
1997.
20.
Huang, LE,
Willmore WG,
Gu J,
Goldberg MA,
and
Bunn HF.
Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling.
J Biol Chem
274:
9038-9044,
1999.
21.
Jernigan, NL,
O'Donaughy TL,
and
Walker BR.
Correlation of HO-1 expression with onset and reversal of hypoxia-induced vasoconstrictor hyporeactivity.
Am J Physiol Heart Circ Physiol
281:
H298-H307,
2001.
22.
Kalaidjieva, V.
Development of bone marrow erythroblastic islands in hypoxic rats with intact or damaged kidney tubules.
Methods Find Exp Clin Pharmacol
20:
841-848,
1998.
23.
Kalaidjieva, V.
Modulation of erythropoiesis in rat bone marrow erythroblastic islands by cyclooxygenase inhibition.
Gen Pharmacol
32:
423-428,
1999.
24.
Kalaidjieva, V,
and
Iliev Z.
Relationship between absolute and relative hematocrit changes and bone marrow response in rats.
Folia Med (Plovdiv)
41:
56-61,
1999.
25.
Kambe, T,
Tada-Kambe J,
Kuge Y,
Yamaguchi-Iwai Y,
Nagao M,
and
Sasaki R.
Retinoic acid stimulates erythropoietin gene transcription in embryonal carcinoma cells through the direct repeat of a steroid/thyroid hormone receptor response element half-site in the hypoxia-response enhancer.
Blood
96:
3265-3271,
2000.
26.
Kearns, CF,
Lenhart JA,
and
McKeever KH.
Cross reactivity between human erythropoietin antibody and horse erythropoietin.
Electrophoresis
21:
1454-1457,
2000.
27.
Kim, HP,
Lee JY,
Jeong JK,
Bae SW,
Lee HK,
and
Jo I.
Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor- localized in caveolae.
Biochem Biophys Res Commun
263:
257-262,
1999.
28.
Kimura, H,
Weisz A,
Ogura T,
Hitomi Y,
Kurashima Y,
Hashimoto K,
D'Acquisto F,
Makuuchi M,
and
Esumi H.
Identification of hypoxia-inducible factor-1 (HIF-1) ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide.
J Biol Chem
276:
2292-2298,
2000.
29.
Kramer, BK,
Bucher M,
Sandner P,
Ittner KP,
Riegger GA,
Ritthaler T,
and
Kurtz A.
Effects of hypoxia on growth factor expression in the rat kidney in vivo.
Kidney Int
51:
444-447,
1997.
30.
Legan, SJ,
Coon GA,
and
Karsch FJ.
Role of estrogen as initiator of daily LH surges in the ovariectomized rat.
Endocrinology
96:
50-56,
1975.
31.
Lemke, SL,
Mayura K,
Reeves WR,
Wang N,
Fickey C,
and
Phillips TD.
Investigation of organophilic montmorillonite clay inclusion in zearalenone-contaminated diets using the mouse uterine weight bioassay.
J Toxicol Environ Health
62:
243-258,
2001.
32.
MacRitchie, AN,
Jun SS,
Chen Z,
German Z,
Yuhanna IS,
Sherman TS,
and
Shaul PW.
Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium.
Circ Res
81:
355-362,
1997.
33.
Maitani, Y,
Moriya H,
Shimoda N,
Takayama K,
and
Nagai T.
Distribution characteristics of entrapped recombinant human erythropoietin in liposomes and its intestinal absorption in rats.
Int J Pharm
185:
13-22,
1999.
34.
Masuda, S,
Kobayashi T,
Chikuma M,
Nagao M,
and
Sasaki R.
The oviduct produces erythropoietin in an estrogen-regulated manner.
Am J Physiol Endocrinol Metab
278:
E1038-E1044,
2000.
35.
Moore, LG,
McMurtry IF,
and
Reeves JT.
Effects of sex hormones on cardiovascular and hematologic responses to chronic hypoxia in rats.
Proc Soc Exp Biol Med
158:
658-662,
1978.
36.
O'Donaughy, TL,
and
Walker BR.
Renal vasodilatory influence of endogenous carbon monoxide in chronically hypoxic rats.
Am J Physiol Heart Circ Physiol
279:
H2908-H2915,
2000.
37.
Ohigashi, T,
Mallia CS,
McGary E,
Scandurro AB,
Rondon I,
Fisher JW,
and
Beckman BS.
Protein kinase C- protein expression is necessary for sustained erythropoietin production in human hepatocellular carcinoma (Hep3B) cells exposed to hypoxia.
Biochim Biophys Acta
1450:
109-118,
1999.
38.
Ou, LC,
Salceda S,
Schuster SJ,
Dunnack LM,
Brink-Johnsen T,
Chen J,
and
Leiter JC.
Polycythemic responses to hypoxia: molecular and genetic mechanisms of chronic mountain sickness.
J Appl Physiol
84:
1242-1251,
1998.
39.
Padilla-Banks, E,
Jefferson WN,
and
Newbold RR.
The immature mouse is a suitable model for detection of estrogenicity in the uterotropic bioassay.
Environ Health Perspect
109:
821-826,
2001.
40.
Peschle, C,
Rappaport IA,
Sasso GF,
Condorelli M,
and
Gordon AS.
The role of estrogen in the regulation of erythropoietin production.
Endocrinology
92:
358-362,
1973.
41.
Pololi-Anagnostou, L,
Schade S,
and
Anagnostou A.
The effect of estrogens on erythroid stem cells in polycythemic mice.
Mol Cell Endocrinol
24:
73-84,
1981.
42.
Porst, M,
Hartner A,
Krause H,
Hilgers KF,
and
Veelken R.
Inducible nitric oxide synthase and glomerular hemodynamics in rats with liver cirrhosis.
Am J Physiol Renal Physiol
281:
F293-F299,
2001.
43.
Reckelhoff, JF,
Hennington BS,
Moore AG,
Blanchard EJ,
and
Cameron J.
Gender differences in the renal nitric oxide (NO) system: dissociation between expression of endothelial NO synthase and renal hemodynamic response to NO synthase inhibition.
Am J Hypertens
11:
97-104,
1998.
44.
Resta, TC,
Chicoine LG,
Omdahl JL,
and
Walker BR.
Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia.
Am J Physiol Heart Circ Physiol
276:
H699-H708,
1999.
45.
Resta, TC,
Gonzales RJ,
Dail WG,
Sanders TC,
and
Walker BR.
Selective upregulation of arterial endothelial nitric oxide synthase in pulmonary hypertension.
Am J Physiol Heart Circ Physiol
272:
H806-H813,
1997.
46.
Resta, TC,
Kanagy NL,
and
Walker BR.
Estradiol induced attenuation of pulmonary hypertension is not associated with altered eNOS expression.
Am J Physiol Lung Cell Mol Physiol
280:
L88-L97,
2001.
47.
Revillion, F,
Pawlowski V,
Hornez L,
and
Peyrat JP.
Glyceraldehyde-3-phosphate dehydrogenase gene expression in human breast cancer.
Eur J Cancer
36:
1038-1042,
2000.
48.
Robertson, JA,
Bhattacharyya S,
and
Ing NH.
Tamoxifen up-regulates oestrogen receptor-, c-fos, and glyceraldehyde 3-phosphate-dehydrogenase mRNAs in ovine endometrium.
J Steroid Biochem Mol Biol
67:
285-292,
1998.
49.
Rupnow, HL,
Phernetton TM,
Shaw CE,
Modrick ML,
Bird IM,
and
Magness RR.
Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS.
Am J Physiol Heart Circ Physiol
280:
H1699-H1705,
2001.
50.
Sandau, KB,
Fandrey J,
and
Brune B.
Accumulation of HIF-1 under the influence of nitric oxide.
Blood
97:
1009-1015,
2001.
51.
Sandau, KB,
Zhou J,
Kietzmann T,
and
Brune B.
Regulation of the hypoxia-inducible factor 1 by the inflammatory mediators nitric oxide and tumor necrosis factor- in contrast to desferroxamine and phenylarsine oxide.
J Biol Chem
276:
39805-39811,
2001.
52.
Sasaki, R,
Masuda S,
and
Nagao M.
Erythropoietin: multiple physiological functions and regulation of biosynthesis.
Biosci Biotechnol Biochem
64:
1775-1793,
2000.
53.
Sasaki, R,
Masuda S,
and
Nagao M.
Pleiotropic functions and tissue-specific expression of erythropoietin.
News Physiol Sci
16:
110-113,
2001.
54.
Schuster, SJ,
Badiavas EV,
Costa-Giomi P,
Weinmann R,
Erslev AJ,
and
Caro J.
Stimulation of erythropoietin gene transcription during hypoxia and cobalt exposure.
Blood
73:
13-16,
1989.
55.
Shanks, JH,
Hill CM,
Lappin TR,
and
Maxwell AP.
Localization of erythropoietin gene expression in proximal renal tubular cells detected by digoxigenin-labelled oligonucleotide probes.
J Pathol
179:
283-287,
1996.
56.
Shaul, PW.
Ontogeny of nitric oxide in the pulmonary vasculature.
Semin Perinatol
21:
381-392,
1997.
57.
Sogawa, K,
Numayama-Tsuruta K,
Ema M,
Abe M,
Abe H,
and
Fujii-Kuriyama Y.
Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia.
Proc Natl Acad Sci USA
95:
7368-7373,
1998.
58.
Suzuma, I,
Mandai M,
Takagi H,
Suzuma K,
Otani A,
Oh H,
Kobayashi K,
and
Honda Y.
17-Estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells.
Invest Ophthalmol Vis Sci
40:
2122-2129,
1999.
59.
Tatsumi, K,
Hannhart B,
Pickett CK,
Weil JV,
and
Moore LG.
Influences of gender and sex hormones on hypoxic ventilatory response in cats.
J Appl Physiol
71:
1746-1751,
1991.
60.
Tatsumi, K,
Pickett CK,
Jacoby CR,
Weil JV,
and
Moore LG.
Role of endogenous female hormones in hypoxic chemosensitivity.
J Appl Physiol
83:
1706-1710,
1997.
61.
Tenenhouse, HS.
Vitamin D metabolism and phosphate transport in developing kidney: effect of diet and mutation.
Pediatr Nephrol
2:
171-175,
1988.
62.
Todorov, V,
Gess B,
Godecke A,
Wagner C,
Schrader J,
and
Kurtz A.
Endogenous nitric oxide attenuates erythropoietin gene expression in vivo.
Pflügers Arch
439:
445-448,
2000.
63.
Tojo, A,
Kimoto M,
and
Wilcox CS.
Renal expression of constitutive NOS and DDAH: separate effects of salt intake and angiotensin.
Kidney Int
58:
2075-2083,
2000.
64.
Vagnoni, KE,
Shaw CE,
Phernetton TM,
Meglin BM,
Bird IM,
and
Magness RR.
Endothelial vasodilator production by uterine and systemic arteries. III. Ovarian and estrogen effects on NO synthase.
Am J Physiol Heart Circ Physiol
275:
H1845-H1856,
1998.
65.
Walker, BR,
Resta TC,
and
Nelin LD.
Nitric oxide-dependent pulmonary vasodilation in polycythemic rats.
Am J Physiol Heart Circ Physiol
279:
H2382-H2389,
2000.
66.
Wen, D,
Boissel JP,
Tracy TE,
Gruninger RH,
Mulcahy LS,
Czelusniak J,
Goodman M,
and
Bunn HF.
Erythropoietin structure-function relationships: high degree of sequence homology among mammals.
Blood
82:
1507-1516,
1993.
67.
Yasuda, Y,
Masuda S,
Chikuma M,
Inoue K,
Nagao M,
and
Sasaki R.
Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis.
J Biol Chem
273:
25381-25387,
1998.
68.
Yoshioka, K,
and
Fisher JW.
Nitric oxide enhancement of erythropoietin production in the isolated perfused rat kidney.
Am J Physiol Cell Physiol
269:
C917-C922,
1995.
69.
Zhong, H,
and
Simons JW.
Direct comparison of GAPDH, -actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia.
Biochem Biophys Res Commun
259:
523-526,
1999.
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