AJP - Heart Watch the video to learn how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 293: H2894-H2903, 2007. First published August 24, 2007; doi:10.1152/ajpheart.01230.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/H2894    most recent
01230.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Østergaard, L.
Right arrow Articles by Simonsen, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Østergaard, L.
Right arrow Articles by Simonsen, U.

Diminished NO release in chronic hypoxic human endothelial cells

Louise Østergaard,1 Edgaras Stankevicius,1 Malene R. Andersen,3,4 Yvonne Eskildsen-Helmond,1 Thomas Ledet,2 Michael J. Mulvany,1 and Ulf Simonsen1

1Department of Pharmacology, University of Aarhus, Aarhus; 2Research Laboratory for Biochemical Pathology, Aarhus Sygehus, Aarhus; 3Research Laboratory of Obstetrics and Gynecology, Skejby Hospital, University of Aarhus, Aarhus; and 4Research Laboratory, Department of Clinical Biochemistry, Gentofte Hospital, University of Copenhagen, Hellerup, Denmark

Submitted 8 November 2006 ; accepted in final form 12 August 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study addressed whether chronic hypoxia is associated with reduced nitric oxide (NO) release due to decreased activation of endothelial NO synthase (eNOS). Primary cultures of endothelial cells from human umbilical veins (HUVECs) were used and exposed to different oxygen levels for 24 h, after which NO release, intracellular calcium, and eNOS activity and phosphorylation were measured after 24 h. Direct measurements using a NO microsensor showed that in contrast to 1-h exposure to 5% and 1% oxygen (acute hypoxia), histamine-evoked (10 µM) NO release from endothelial cells exposed to 5% and 1% oxygen for 24 h (chronic hypoxia) was reduced by, respectively, 58% and 40%. Furthermore, chronic hypoxia also lowered the amount and activity of eNOS enzyme. The decrease in activity could be accounted for by reduced intracellular calcium and altered eNOS phosphorylation. eNOS Ser1177 and eNOS Thr495 phosphorylations were reduced and increased, respectively, consistent with lowered enzyme activity. Akt kinase, which can phosphorylate eNOS Ser1177, was also decreased by hypoxia, regarding both total protein content and the phosphorylated (active) form. Moreover, the protein content of beta- actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. In conclusion, chronic hypoxia in HUVECs reduces histamine-induced NO release as well as eNOS expression and activity. The decreased activity is most likely due to changed eNOS phosphorylation, which is supported by decreases in Akt expression and phosphorylation. By reducing NO, chronic hypoxia may accentuate endothelial dysfunction in cardiovascular disease.

endothelial nitric oxide synthase; Akt; actin; endothelium; hypoxia

HYPOXIA is common in patients with cardiopulmonary disease (22), and chronic hypoxia during the course of pregnancy is one of the common insults thought to be associated with increased risk of preeclampsia and fetal intrauterine growth restriction (37). Even though the endothelial cell (EC) layer is relatively tolerant to low oxygen tensions (20), many of the effects of chronic hypoxia are thought to take place through alterations in EC signaling. This can affect the release of vasoactive products such as nitric oxide (NO), which contributes not only to the local regulation of vascular smooth muscle tone (54) but also regulates cell proliferation (19). Endothelium-derived NO was suggested to contribute to acute hypoxic vasodilation in human umbilical vein ring segments (32), whereas NO synthase (NOS) inhibition hardly influenced flow-evoked endothelium-dependent vasodilation in porcine coronary resistance arteries (21). In chronic hypoxia, in vivo studies have suggested that NO-dependent vasodilatation is decreased in the rat aorta and in uterine arteries from pregnant guinea pigs (31, 56), whereas it appears to be increased in pregnant ovine uterine arteries (58) and small mesenteric arteries (13) and in fetal guinea pig hearts (52). Hypoxia was reported to increase NO end products in pregnant ovine uterine arteries (58), and direct measurements with microsensors in mesenteric arteries also suggested that NO was increased (13), whereas fluorescence measurements revealed that NO was decreased in pulmonary arteries from chronic hypoxic rats (35). Thus, the response to hypoxia appears to depend on the vascular bed involved, degree and duration of hypoxia, gestational state, and in vivo additional indirect effects caused by, e.g., changes in blood flow and shear stress influencing the endothelial NO system.

Measurements of the direct effect of hypoxia on ECs in culture, however, also revealed contrasting effects of chronic hypoxia: hypoxia was found to increase the formation of NO end products in cultured coronary ECs (7, 25, 44, 59), whereas they were decreased in bovine aortic and pulmonary ECs and human umbilical vein ECs (HUVECs) (28, 57); others reported no change in HUVECs (10). Measurments of the conversion of L-arginine to L-citrulline suggested that endothelial NOS (eNOS) activity decreases in bovine and human ECs exposed to chronic hypoxia (3, 50). In regard to the expression of eNOS protein and mRNA in cultured ECs, it has been reported to be increased after short-term (7, 21, 44) or chronic (3) hypoxia, whereas chronic hypoxia has been reported to decrease protein and mRNA expression in HUVECs (32), bovine pulmonary arterial cells (28, 39), and human pulmonary arterial and saphenous vein ECs (50). Thus, the EC type involved seems to influence the response to chronic hypoxia, but it is also apparent that several of the studies have only considered part of the NO pathway.

In the present study, we sought to provide a better framework for understanding the cardiovascular consequences of chronic hypoxia in humans by determining the effect of chronic hypoxia on several aspescts of the L-arginine/NO pathway, from the activation of eNOS to the release of NO. We used primary cultures of HUVECs and exposed them to 5% or 1% oxygen for 24 h. NO release was measured directly with the use of NO-sensitive microsensors. Biochemical experiments were performed to determine the degree to which changes in NO production were related to alterations in eNOS expression, activity, and phosphorylation. The effectiveness of the hypoxia was determined by the effect on hypoxia-inducible factor (HIF), known to be stabilized by hypoxia (42). The role of HIF in the biochemical changes seen was determined with the use of CoCl2, known also to stabilize HIF (60). We hypothesized that chronic hypoxia is associated with reduced NO release due to decreased activation of eNOS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Study participants. Pregnant women attending routine antenatal care in the period of November 2004 to June 2006 at the Department of Obstetrics and Gynecology, Skejby Hospital, were invited to participate in the study. The women included were nonsmoking, normotensive, had normal cholesterol levels, and did not have preeclampsia, diabetes mellitus, or a family history of premature vascular disease; none were on regular medication.

Written consent was obtained from those who agreed to participate (58 women total). The investigation conformed to the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee of the University of Aarhus (Reference No. 20040154).

Cell isolation and culture. Umbilical cords were obtained immediately after delivery and transported from the Department of Obstetrics and Gynecology to the Department of Pharmacology, where HUVECs were isolated as previously described (40). Briefly, veins were first rinsed in DMEM (Sigma Aldrich, St. Louis, MO) to remove as much blood as possible. Approximately 10 ml of a 0.1% collagenase solution were flushed through the vein, and the umbilical cord was closed at both ends by clamps. After the umbilical cord had been incubated for 1 h at 37°C, the collagenase solution was washed out with DMEM, and 20 ml of stop medium (DMEM with 10% FCS) were added. Cells were spun down for 8 min at 2,100 rpm, the supernatant was discarded, and the pellet was resuspended in growth medium containing 2% serum, EGF, hydrocortisone, VEGF, FGF, IGF, ascorbic acid, heparin, amphotericin B, and gentamicin (Endothelial Cell Growth Medium 2 with supplement pack, Promocell, Heidelberg, Germany). Cells were then plated out in gelatine-coated flasks or dishes and allowed to adhere overnight. Growth medium was changed every second day, and cells were passaged with 0.25% trypsin when confluent. In all experiments, cells were used at passages 2 or 3. The EC phenotype was confirmed using phase-contrast microscopy (i.e., cuboidal, cobblestone-appearing monolayer of cells) and positive immunofluorescence staining with antibodies against von Willebrand factor (40). Confluent monolayers of ECs were established and incubated for 24 h at 5% or 1% oxygen in an air-tight chamber placed in a heating cabinet. The low oxygen concentration was obtained by flushing the chamber with a gas mixture consisting of 95% N2-5% CO2, and the oxygen concentration was continuously monitored using an oxygen minisensor electrode (Unisense, Aarhus, Denmark). The precise concentration at the surface of the cells was also measured during normoxia (data not shown) and was similar to the oxygen concentration in the media after 24 h compared with the concentration of oxygen measured in the incubation chamber. Cobalt stabilizes HIF (60), and, to investigate the involvement of HIF, CoCl2 was dissolved in growth medium to a final concentration of 10 mM; cells were incubated with this medium for 4 h at normal oxygen tension. After the hypoxic treatment, a cell count was performed.

NO measurements in cell culture. ECs were grown until near confluence on gelatine-covered glass slides (15 mm) and then incubated for 24 h under either 5% or 1% oxygen. Slides were then placed in a bath with physiological salt solution (PSS) containing 1.6 mM Ca2+. With the use of a micromanipulator (Leitz, Wetzlar, Germany), a NO-sensitive microsensor with a diameter of 30–50 µm (ISONOP30, World Precision Instruments, Stevenage, UK) was placed 5 µm above the slide for NO measurements. Experiments were performed at 37°C in 21%, 5%, and 1% oxygen in the bath. The calibration was carried out with the use of a NO gas solution as previously described (43, 55). Sensitivity was checked before and after each experimental protocol and, in all experiments, remained unchanged. To test the selectivity of the microsensors, a lack of response to sodium nitrite up to 10 µM was taken as evidence for the intact coating of the microsensor. NO release was measured at baseline and in response to 0.1, 1, and 10 µM histamine. To address the effect of acute hypoxia on the release of NO from human ECs, HUVECs were exposed to 21%, 5%, or 1% oxygen for 1 h, and increases in NO concentration evoked by histamine were measured as described above.

There was an increase in sensitivity of the NO microsensor when it was used for measurements at low oxygen tensions (Fig. 1). During calibration of the microsensor, by the addition of increasing amounts of NO gas, it was observed that low oxygen concentrations resulted in a higher signal from the microsensor. The highest signal intensity was seen with 1% oxygen, and it was significant lower at 5% and 21% oxygen.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 1. Calibration and signal sensitivity of the nitric oxide (NO) microsensor. A: original representative traces showing the measurement of NO during calibration at 21%, 5%, and 1% oxygen. B: summary of experiments (n = 5) showing that the sensitivity toward NO was highest at 1% oxygen and significantly lower at 5% and 21% oxygen (*P < 0.05). There were no significant differences between 5% and 21% oxygen.

 
Measurement of eNOS activity. From the same umbilical cord, ECs were grown in 6-cm petri dishes and incubated under 21%, 5%, or 1% oxygen. Immediately after the hypoxic incubation, cells were washed twice in PBS-Tween 20 (PBS-T; containing 150 mM NaCl, 50 mM NaH2PO4, and 0.05% Tween 20), scraped off in 600 µl of 0.05 M Tris-buffered saline (TBS; containing 20 mM Tris·HCl and 137 mM NaCl; pH 7.4), and divided into Eppendorf tubes containing 250 µl for the citrulline assay and 50 µl for the cell count. All tubes were frozen in liquid nitrogen and stored at –80°C until assayed. The activity of eNOS was determined by the conversion of L-[14C]arginine to L-[14C]citrulline with minor modifications of the methods previously described (2). First, cell numbers in the sample were determined by cell counting in a Burker-Türk counting chamber at the light microscopic level to express eNOS activity per million HUVECs. Second, HUVECs were homogenized by six cycles of freeze/thawing, and 25 µl of homogenate were incubated for 30 min at 37°C in a Tris reaction buffer containing L-[14C]arginine, calcium, and the cofactors needed for eNOS activation [calmodulin, flavin adenine dinucleotide, beta-NADPH, and tetrahydrobiopterin (final volume: 150 µl)]. L-[14C]citrulline was isolated by column chromatography and quantified by liquid scintillation counting. eNOS activity was calculated from the mean formation of L-[14C]citrulline per minute and mean numbers of HUVECs in the sample (picomoles of L-citrulline per minute per 106 HUVECs).

The intra-assay coefficient of variation (CV) was 6% (n = 6) and the interassay CV was 5% (n = 6) when measured over a period of 4 wk. This was based on ECs from thoracic pig aortas (2).

Measurement of eNOS concentration. A human eNOS Quantikine Immunoassay kit (R&D Systems, Abingdon, UK) was used for quantification of the eNOS concentration. The assay procedure of the kit was followed, but only 50 µl of cell homogenate in Tris buffer (final volume: 100 µl) were added per well. All samples were assayed in duplicate, and mean eNOS concentrations were calculated relative to the numbers of HUVECs (ng eNOS/106 HUVECs). For controls, recombinant human eNOS standards were used, and the intra-assay CV was 5% (n = 6).

Immunoblot analysis. Immediately after hypoxic or CoCl2 incubation, cells were washed twice in PBS-T, frozen in liquid nitrogen (still in the dish), and stored at –80°C until used. Cells were then scraped off directly in sample buffer [8.5% glycerine, 2% SDS, 62.7 mM Tris·HCl (pH 6.8), 20 mM DTT, and 0.002% bromophenol blue] using a rubber policeman and boiled for 15 min. Protein concentrations were measured using a noninterfering protein assay kit (Calbiochem). Samples, containing an equal amount of protein, were loaded on 4–12% polyacrylamide bis-Tris criterion gels (Bio-Rad Laboratories, Hercules, CA), and protein separation was carried out in the Criterion Electrophoresis System (Bio-Rad Laboratories, Hercules, CA) using XT MOPS as the running buffer. Proteins were blotted onto a polyvinylidine difluoride membrane for 1 h at 100 mV. Membranes were incubated as follows: first, they were blocked for 1 h in 5% milk, and the primary antibody was then added in the right dilution (in 5% milk). Membranes were left rotating at 4°C overnight. The next day, membranes were washed three times for 10 min in TBS-Tween 20 (TBS-T) or PBS-T before secondary antibodies were added (anti-mouse or anti-rabbit antibodies; also in 5% milk). After being incubated for 1 h at room temperature, membranes were again washed in TBS-T or PBS-T and then developed using an enhanced chemiluminescence detection system (ECL or ECL+, Amersham Biosciences). Bands were visualized on film (Amersham Bioscience) and scanned with a GS-710 Imaging Densitometer (Bio-Rad, U.S.A.) using the Quantity One software package, and intensities of the bands were evaluated using ImageQuant software (GE Healthcare).

Antibodies. The antibodies used were anti-human HIF-1{alpha} clone 54 (BD Biosciences, San Jose, CA), anti-human eNOS (Abcam, Cambridge, UK), anti-human eNOS Ser1177 (Upstate, Lake Placid, NY), anti-human eNOS Thr495 (Upstate), anti-human Akt (Cell Signaling, Danvers, MA), anti-human Akt Ser473 (Cell Signaling), and anti-human beta-actin clone AC-15 (Abcam).

Calcium measurements. Confluent HUVECs (passages 2–3) were incubated under 21%, 5%, or 1% oxygen for 23 h, loaded with fura-2 AM, and then incubated an additional 1 h under the specified oxygen concentration before being mounted in a perfusion chamber (Warner Instruments). With the use of a Deltaram lamp, fura-2-loaded cells were excited at 340, 360, and 380 nm, and the light passing through a 510-nm filter was captured by an intensified charge-coupled device camera operated using the IC300 digital imaging system (PTI) for the three wavelengths of 340, 360, and 380 nm. Ratio images of 340/380 nm were used for measurements of increases in the intracellular calcium concentration ([Ca2+]i). To investigate the change in calcium concentration induced by histamine upon normoxia and hypoxia, ECs were perfused first with PSS and then with histamine (1 µM). By the end of the experiments, the maximal increase in calcium was obtained by the addition of 1 µM ionomycin. All solutions were bubbled with nitrogen with 5% CO2 until the desired oxygen concentration (5% or 1%) was reached (measured using a Clark oxygen electrode).

Data analysis. Values from 5% and 1% hypoxia and from CoCl2 treatment were normalized against their respective control value (100%), and results are expressed as mean values ± SE; n refers to the number of umbilical veins used for each experiment. Unless otherwise stated, differences were statistically evaluated by one-way ANOVA followed by a Student-Newman-Keuls posttest. Statistical calculations were made using GraphPad Prism version 4.0 (Institute for Scientific Information, San Diego, CA). Differences were considered significant when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HIF-1{alpha} stabilization under hypoxic incubation. In EC cultures from six different umbilical cords kept at 21% oxygen, there were 800 ± 60 cells/µl present, 1,290 ± 60 cells/µl with 5% oxygen, and 840 ± 20 cells/µl after 1% oxygen. The concentration of oxygen in the air-tight chamber was continuously measured with an oxygen electrode during the entire 24 h of incubation to verify that the oxygen tension was kept within 0.5% of the desired level (data not shown). In these conditions, hypoxia (5% and 1% oxygen) and CoCl2 stabilized HIF-1{alpha}, as visualized by immunoblot analysis (Fig. 2).


Figure 2
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 2. Stabilization of hypoxia-inducible factor (HIF)-1{alpha} at hypoxic oxygen concentrations. A: immunoblots showing the stabilization of HIF-1{alpha} in human umbilical vein endothelial cells (HUVECs) under both levels of hypoxia as well as with incubation with CoCl2. B: summary of experiments (n = 6–17) showing the amount of HIF-1{alpha} after normoxia and hypoxia (5% and 1% oxygen) and CoCl2 treatment. All values are normalized to CoCl2. Increases in protein content from 21% to 5% oxygen and between 1% oxygen and CoCl2 treatment were significant (*P < 0.001).

 
Histamine-induced release of NO in cells exposed to hypoxia. In HUVECs exposed to 21% oxygen, histamine induced a concentration-dependent release of NO (Fig. 3A). The histamine-induced NO release was decreased markedly in cells exposed to 5% and 1% oxygen for 24 h (Fig. 3B). In parallel experiments with ECs exposed to 21% oxygen for 24 h, histamine (1 µM) increased the NO concentration with 21.9 ± 5.6 nM in the absence and with 2.0 ± 0.6 nM in the presence of the NOS inhibitor asymmetric dimethylarginine (300 µM) (cells from 5 umbilical cords; data not shown).


Figure 3
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 3. Histamine-induced release of NO in HUVECs exposed to hypoxia. A: representative trace of the measurement of NO release in cells exposed to the indicated oxygen tensions for 24 h. The microsensor was first allowed to stabilize under 21%, 5%, or 1% oxygen, and the release of NO in response to 3 concentrations of histamine was measured (0.1, 1.0, and 10 µM). B: average increases in NO concentration induced by histamine in cells exposed to 21%, 5%, and 1% oxygen for 24 h. The release of NO was decreased in HUVECs exposed to 5% and 1% oxygen for 1 h. C: average increases in NO concentration induced by histamine in cells exposed to 21%, 5%, and 1% oxygen for 1 h. NO concentration was increased in cells exposed to 5% oxygen, whereas it was unaltered in cells exposed to 1% oxygen compared 21% oxygen. Values are means ± SE; n = 4–5 experiments (1 experiment for each subject). Differences in responses were evaluated by two-way ANOVA. *P < 0.05 vs. histamine-evoked NO release at normoxia (21% oxygen); #P < 0.05 vs. histamine-evoked NO release at 5% oxygen. Increases in the NO concentration were dependent on the histamine concentration applied.

 
In cells exposed to 5% oxygen for 1 h, histamine-evoked increases in the NO concentration were significantly increased compared with cells exposed to 21% oxygen for 1 h, whereas histamine induced similar increases in ECs exposed to 1% and 21% oxygen for 1 h (Fig. 3C).

Effect of hypoxia on eNOS activity and protein levels. The activity of the eNOS enzyme was determined by measuring the conversion of radioactively labeled L-arginine to L-citrulline. The eNOS activity was reduced in response to 5% and 1% oxygen compared with 21% oxygen (Fig. 4A). Thus, the eNOS activity was significantly lowered to 60 ± 9% and 55 ± 7% at 5% and 1% oxygen, respectively. The same samples were also used for measuring the protein level of eNOS by ELISA, which showed that the protein level was reduced in the same manner as the activity (Fig. 4B). Thus, the amount of eNOS protein was decreased to 63.2 ± 7.5% at 5% oxygen (P < 0.05 vs. normoxia) and even further to 39.4 ± 6.2% at 1% oxygen (P < 0.05 vs. 21% and 5% oxygen). In contrast, immunoblot analysis showed the HIF activator CoCl2 (100 µM) increased eNOS content by 136 ± 11% (P < 0.05 vs. control, n = 7; data not shown).


Figure 4
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 4. Effect of hypoxia on endothelial NO synthase (eNOS) activity and concentration. The rate of conversion of L-[14C]arginine to L-[14C]citrulline (A) and eNOS concentration (B) were significantly lowered during hypoxia. The eNOS activity and concentration are expressed relative to the control value (100%). Values are means ± SE of 7 experiments (1 experiment for each subject). *P < 0.05 vs. 21% oxygen.

 
eNOS phosphorylation. The relative amount of eNOS phosphorylated at the Ser1177 residue, as determined using immunoblot analysis, was decreased in response to hypoxia (Fig. 5A). This was also the case after CoCl2 incubation. In HUVECs from three umbilical cords kept at 1% oxygen for 1 h, we moreover measured eNOS expression and eNOS Ser1177 phosphorylation and found that it was unchanged compared with cells kept at 21% oxygen (results not shown). The phosphorylation at eNOS Thr495 was, in contrast to eNOS Ser1177, increased in cells exposed to 5% and 1% oxygen for 24 h (Fig. 5B). During CoCl2 incubation, the relative amount of eNOS Thr495 phosphorylation was not changed.


Figure 5
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 5. Hypoxia and CoCl2-induced alterations in eNOS phosphorylation at Ser1177 and Thr495. A: phosphorylation of Ser1177 was significantly lowered with both hypoxia and CoCl2 treatment. B: phosphorylation at Thr495, on the other hand, was increased during hypoxia and slightly, but significantly, decreased during CoCl2 incubation. The amount of phosphorylated eNOS was expressed relative to the total amount of eNOS protein. Values are means ± SE; n = 6–12 experiments (1 experiment from each subject). *P < 0.05 vs. normoxia. Differences from 5% to 1% were not significant. Note that the scale in B is different from the scale in other figures.

 
Akt expression and phosphorylation. Akt is considered the main kinase responsible for the phosphorylation of eNOS Ser1177 (12, 17). Akt protein content was reduced to 64 ± 6% and 45 ± 8% for 5% and 1% oxygen, respectively, whereas CoCl2 treatment did not change Akt protein content (Fig. 6A). The same regulation was also seen for the phosphorylated/active form of Akt. Thus, the decrease in phosphorylated active Akt was, respectively, 77 ± 5% and 60 ± 7% for 5% and 1% oxygen (Fig. 6B).


Figure 6
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 6. Akt expression and phosphorylation. Akt protein content (A) and phosphorylation of Akt (Akt-P; B) were decreased at both 5% and 1% oxygen, whereas CoCl2 treatment did not change the protein content. Values are means ± SE of 7–18 experiments (1 experiment for each subject). *P < 0.05 vs. normoxia; #P < 0.05 vs. 5% oxygen.

 
Effect of hypoxia on beta-actin expression. The amount of the SDS-soluble fraction of the structural protein beta-actin was approximately halved after the exposure to 5% and 1% oxygen (Fig. 7). The incubation with CoCl2 did not change beta-actin protein content.


Figure 7
View larger version (27K):
[in this window]
[in a new window]

  		Fig. 7. Effect of hypoxia on beta-actin expression. Top: representative immunoblots for actin showing that hypoxia downregulated beta-actin. Bottom: values are means ± SE of 8–15 experiments (1 experiment for each subject). *P < 0.05 vs. normoxia. There were no differences in the amount of beta-actin in HUVECs exposed to 5% vs. 1% oxygen. The HIF activator CoCl2 did not change the beta-actin content.

 
Effect of chronic hypoxia on EC calcium levels. Loading of ECs with fura-2 was comparable, since fluorescence measurements at the isobestic wavelength of 360 nm were not different; photometric counts were 114,925 ± 6,289 counts/s in normoxia, 113,075 ± 3,630 counts/s at 5% oxygen, and 110,240 ± 5,082 counts/s at 1% oxygen. Baseline calcium levels expressed as the ratio of emission intensities at the two excitation wavelengths (340 vs. 380 nm) in cells exposed for 24 h to 21%, 5%, and 1% oxygen were, respectively, 0.130 ± 0.015, 0.072 ± 0.012 (P < 0.05 vs. 21% oxygen, n = 5), and 0.022 ± 0.017 (P < 0.05 vs. 21% oxygen, n = 5). Histamine (1 µM)-evoked increases in the fura-2 ratio in EC calcium were significantly less pronounced at 5 and 1% oxygen compared with 21% oxygen (Fig. 8A). Calcium ionophore ionomycin (1 µM)-evoked increases in calcium levels were less in cells exposed to 1% oxygen (Fig. 8B).


Figure 8
View larger version (12K):
[in this window]
[in a new window]

  		Fig. 8. Measurements of EC calcium levels in HUVECs with the use of fura-2. Increases in EC calcium levels were evoked by histamine (1 µM; A) and ionomycin (1 µM; B). Changes in fura-2 fluorescence were expressed as increases in the ratio of emission intensities at the two excitation wavelengths (340 vs. 380 nm) [{Delta}(F340/F380)]. Results are means ± SE of cells from 5–6 umbilical cords. *P < 0.05 vs. normoxia.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main finding of the present study was that NO release from ECs was diminished after 24-h incubation with 5% and 1% oxygen. This is supported by the findings that the eNOS activity and concentration were lowered by chronic hypoxia. The decreased eNOS activity was probably a consequence of the, respectively, decreased and increased phosphorylations of eNOS at Ser1177 and Thr495, where the altered eNOS phosphorylation at Ser1177 agrees with reduced Akt protein content and phosphorylation.

As mentioned in the Introduction, measurements of NO end products in ECs exposed to chronic hypoxia have suggested either increased (7, 25, 58, 59), decreased (28, 53), or no alterations (10) in nitrite and nitrate levels. Differences in the EC type and duration of hypoxic exposure explain some of these different findings. Moreover, the formation of nitrite and nitrate is cumulative and can result from other processes than degradation of NO (4). With the use of NO-sensitive microsensors, the present study provides direct evidence showing that in contrast to acute lowering of oxygen for 1 h, agonist-induced NO release is successively blunted by lowering the oxygen to 5% and 1% for 24 h. Account was taken of the increased sensitivity of the NO-sensitive microsensors to reduced oxygen tension (55) (Fig. 1), and hence the altered sensitivity of the microsensor does not explain the decreased NO concentration measured in HUVECs exposed to a hypoxic environment for 24 h. Our findings of decreased agonist-evoked NO formation agree with the observation of decreased cGMP formation in primary cultured HUVECs exposed to chronic hypoxia (32). In a previous study (55), we found that histamine-induced NO release from HUVECs is blocked by inhibitors of NOS, and, therefore, our measurements of NO concentration provide direct evidence that the NO concentration is decreased in ECs exposed to chronic hypoxia.

Chronic hypoxia and eNOS expression. The regulation of eNOS expression during hypoxia appears controversial, but it seems that in general, short hypoxic incubations, at oxygen concentrations anywhere between 1% and 5%, result in increased eNOS protein and mRNA expression (6, 21, 29), whereas longer incubations, usually 24 h, result in increased (3) or decreased (28, 39, 50) eNOS protein and/or mRNA expression. In the present study, 24-h incubations with 5% and 1% oxygen also decreased eNOS content per EC, and the decreased expression of eNOS was pronounced, suggesting that it in part explains the decreased release of NO in cells exposed to hypoxia.

To investigate the role of HIF in the regulation of eNOS expression, ECs in the present study were incubated with CoCl2, a known stabilizer of HIF-1{alpha} (60). In contrast to chronic hypoxia, the amount of eNOS was increased with CoCl2 incubation. The latter finding is most likely due to the fact that HIF-2 can bind to a HIF response site (11, 14). Reduced eNOS expression in chronic hypoxia has been ascribed to decreased steady-state mRNA and/or stability (32, 49) or increased proteasomal activation (45). The present study does not exclude that HIF contributes to increased eNOS expression in short-term hypoxia but suggests that other mechanisms lead to the decreased eNOS expression and NO formation in chronic hypoxia.

Chronic hypoxia and eNOS activity. In addition to decreased eNOS expression, hypoxia-induced reduction of eNOS activity could contribute to decreased NO release. In bovine aortic ECs (3) and human saphenous vein ECs (50), chronic hypoxia resulted in reduced eNOS activity. In the present study, chronic hypoxia also reduced activity measured as the conversion of labeled L-arginine to L-citrulline in HUVECs. These findings provide further support to the suggestion that reduced eNOS activity plays an important role for reduced endothelial NO release in chronic hypoxia.

Alterations in substrate availability, endothelial signal transduction, and bioavailability of oxygen may explain the blunted histamine-induced NO release and reduced eNOS activity in HUVECs. The cell medium contained 300 µM L-arginine, which is higher or at least similar to the plasma L-arginine concentration in humans and rats (18, 30), and is therefore an unlikely explanation for the decreased NO release. Oxygen is required for formation of NO from L-arginine (46), but the Km value for eNOS in respect to oxygen is 4 µM (46), and it is therefore not likely that low oxygen by itself explains the decreased NO formation observed in the present study. Decreased bioavailability of NO due to reaction with superoxide is another possibility (4). Thus, short-term exposure to 1% hypoxia was demonstrated to increase mitochondrial free radical generation followed by the formation of IL-6 and increased permeability in the HUVEC monolayer (38). Although our findings suggest that decreased eNOS expression and alterations in eNOS activity contribute to decrease NO formation, further studies are required to clarify whether the formation of radical oxygen species contributes to the decreased eNOS expression and/or NO formation in HUVECs exposed to chronic hypoxia.

Regulation of eNOS. Increases in EC calcium levels and membrane hyperpolarization are coupled to the release of endothelium-derived relaxing factors including NO. Studies of arteries from chronic hypoxic animals have also shown that the EC calcium level is closely related to the release of NO, since the acetylcholine-induced increase in EC calcium and NO concentrations was increased in mesenteric arteries (13) and the carbachol-induced increase in EC calcium and NO concentrations was reduced in pulmonary arteries from chronic hypoxic rats (35). In the present study, the histamine-induced increase in [Ca2+]i and NO concentration was also reduced in ECs exposed chronic hypoxia. These findings suggest that chronic hypoxia alters EC calcium handling and is closely related to the alterations in the release of NO. In severe hypoxia, the ionomycin-induced increase in [Ca2+]i was also blunted, suggesting that intracellular calcium handling and/or activation of calcium-induced calcium release is affected by chronic hypoxia.

eNOS activity is regulated by a variety of factors such as acylation, by its cellular location, and protein-protein interactions such as eNOS-caveolin-1, eNOS-heat shock protein 90 (Hsp90), and eNOS-calmodulin (15). In ovine fetal and neonatal lung microvascular ECs, increasing oxygen at birth was suggested to promote the dissociation of caveolin-1 and eNOS (24), and, in chronic hypoxic rats, tight coupling of caveolin-1 and eNOS resulted in reduced NO formation in pulmonary arteries (35). Efficient NO production requires the binding of Hsp90 to eNOS, and, in porcine coronary artery ECs, short-term hypoxia stimulated Hsp90 binding to eNOS (7), whereas in pulmonary arteries from chronic hypoxic rats, both Hsp90 and calmodulin binding to eNOS was found to be decreased (35). Although our results do not exclude alterations in caveolin-1 and Hsp90 binding to eNOS, the findings of blunted increase in EC calcium levels in the present study suggest that decreased calmodulin-eNOS binding may contribute to the decrease in eNOS activity and NO release observed in hypoxic ECs.

eNOS is also regulated by phosphorylations at multiple sites (15). Various enzymes such as Akt (12, 51), protein kinase A (5), and 5'-AMP-activated protein kinase (8) have been reported to phosphorylate eNOS on Ser1177 residues, resulting in increased NO after different stimuli. Phosphorylation on Thr495, on the other hand, causes a decrease in enzyme activity and NO (8). In the present study, phosphorylation of Ser1177 was decreased and Thr495 increased, suggesting that altered phosphorylation of eNOS explains the decreased enzyme activity and NO output. Moreover, the total amount of Akt as well as the relative amount of the active (phosphorylated) form was reduced after hypoxic incubation, again supporting the previous results. The regulation of phosphatidylinositol 3- kinase (PI3K/Akt) signaling during hypoxia seems to be cell type specific (1), but caspase 3 has been found to cleave Akt (27), and the decrease in Akt phosphorylation can be mediated by, for example, endoplasmic reticulum stress (23) and metalloproteinase 1 (9), all of which are likely to be activated by hypoxia.

The decrease we found in the phosphorylation at Ser1177 with CoCl2 treatment is consistent with a decrease in cyclic nucleotides caused directly by CoCl2. CoCl2 has been shown to reduce the amount of cyclic nucleotides (33) and kinases known to phosphorylate Ser1177, such as PKA and Akt, which are dependent on cAMP. Furthermore, the phosphorylation of Thr495 was not changed with CoCl2 treatment, nor was Akt protein or its phosphorylation. It therefore seems that HIF in chronic hypoxia does not have a significant influence on the regulation of eNOS phosphorylation and the release of NO.

Actin is known to influence eNOS and NO production at several levels (48). This regulation is seen both at the posttranscriptional (41) and posttranslational level (47, 61). In addition, an intact cytoskeleton seems to be important for the phosphorylation and thereby activation of Akt (16), further implicating actin in the regulation of eNOS activity. It has been demonstrated that actin, and especially G-actin, can increase the activity of eNOS (26, 47). Rho kinase, which can induce stress fiber formation and focal adhesions, and which is upregulated during hypoxia, can negatively regulate eNOS at the protein level as well as mRNA stability and activity (47, 50). The actin cytoskeleton can also play a role for keeping eNOS in close proximity to caveolin-1 and also seems able to affect enzyme activity directly (47). In the present study, we found that CoCl2 did not change actin turnover, indicating that HIF is not involved, but, during 5% and 1% hypoxia, the amount of beta-actin was approximately halved compared with normoxia. These findings support the other results in confirming that hypoxia in HUVECs leads to diminished release of NO.

Limitations and conclusions. The degree of hypoxia is of importance for the physiological response. In the present study, we chose to investigate the response to both 5% and 1% oxygen. The oxygen concentration in the human umbilical vein is ~5% (34), and it could thus be argued that 5% oxygen would resemble normoxia rather than hypoxia. However, since cells were cultured at 21% oxygen for ~2 wk before the experiments, it is likely that they have adapted to 21% oxygen as normoxia. This conclusion was supported by our finding that immunoblot analysis demonstrated stabilization of the transcription factor hypoxia HIF-1{alpha} in cells exposed to 1% and 5% oxygen. The stabilization of HIF-1{alpha} under both forms of hypoxia and not under normoxia strongly suggests that cells were reacting to hypoxic incubations.

As mentioned in the Introduction, in vivo the response of the endothelial NO system to chronic hypoxia depends on the vascular bed involved, duration and degree of hypoxia, gestational state, and also blood flow and shear stress. Although the present study cannot reflect all the influence of all these variables, it is focused on the direct effect of chronic hypoxia on the release of NO in primary cultures of human ECs from a large number of subjects and, hence, contributes to the understanding of the mechanisms underlying the decreased formation of NO in these conditions. The decreased production of NO underlies the pathophysiology of a number of important vascular disorders such as atherosclerosis, diabetes, and pulmonary hypertension (34, 55), and, from our results, we would expect that hypoxia-induced reduction of eNOS expression and altered phosphorylation followed by blunted NO release would decrease many of the positive actions of NO that serve to protect the cells and surrounding tissue and, hence, contribute to the development of cardiovascular diseases.

In conclusion, chronic hypoxia in HUVECs reduces the histamine-induced NO release as well as eNOS activity and protein level. The decreased activity is most likely due to a change in eNOS phosphorylations, which is supported by decreases in Akt expression and phosphorylation. Moreover, the protein content of beta-actin, which is known to influence the activity of eNOS, was almost halved by hypoxia, further supporting the fall in eNOS activity. Therefore, based on the present findings in primary cultures of human ECs, it is likely that chronic hypoxia leading to reduced NO release and activity will accentuate endothelial dysfunction associated with cardiovascular disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
L. Østergaard was supported by the Danish Academy of Cardiovascular Research and H. Lundbeck Limited. M. J. Mulvany and U. Simonsen were supported by the Danish Research Council.


   ACKNOWLEDGMENTS
 
We thank Kirsten Zeeberg (Research Laboratory of Obstetrics and Gynecology, Skejby Hospital), Jimmy Weng (Research Laboratory, Department of Clinical Biochemistry, Gentofte Hospital), and Helle Zibrandtsen for technical assistance and Dr. Henrik Vorum for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: U. Simonsen, Dept. of Pharmacology, Faculty of Health Sciences, Univ. of Aarhus, 8000 Aarhus C, Denmark (e-mail: us{at}farm.au.dk)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alvarez-Tejado M, Alfranca A, Aragones J, Vara A, Landazuri MO, del Peso L. Lack of evidence for the involvement of the phosphoinositide 3-kinase/Akt pathway in the activation of hypoxia-inducible factors by low oxygen tension. J Biol Chem 277: 13508–13517, 2002.[Abstract/Free Full Text]
  2. Andersen MR, Stender S. Endothelial nitric oxide synthase activity in aorta of normocholesterolemic rabbits: regional variation and the effect of estrogen. Cardiovasc Res 47: 192–199, 2000.[Abstract/Free Full Text]
  3. Arnet UA, McMillan A, Dinerman JL, Ballermann B, Lowenstein CJ. Regulation of endothelial nitric-oxide synthase during hypoxia. J Biol Chem 271: 15069–15073, 1996.[Abstract/Free Full Text]
  4. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol Cell Physiol 271: C1424–C1437, 1996.[Abstract/Free Full Text]
  5. Butt E, Bernhardt M, Smolenski A, Kotsonis P, Frohlich LG, Sickmann A, Meyer HE, Lohmann SM, Schmidt HH. Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 275: 5179–5187, 2000.[Abstract/Free Full Text]
  6. Chen JX, Lawrence ML, Cunningham G, Christman BW, Meyrick B. HSP90 and Akt modulate Ang-1-induced angiogenesis via NO in coronary artery endothelium. J Appl Physiol 96: 612–620, 2004.[Abstract/Free Full Text]
  7. Chen JX, Meyrick B. Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium. Lab Invest 84: 182–190, 2004.[CrossRef][Web of Science][Medline]
  8. Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE. AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443: 285–289, 1999.[CrossRef][Web of Science][Medline]
  9. Conant K, St Hillaire C, Nagase H, Visse R, Gary D, Haughey N, Anderson C, Turchan J, Nath A. Matrix metalloproteinase 1 interacts with neuronal integrins and stimulates dephosphorylation of Akt. J Biol Chem 279: 8056–8062, 2004.[Abstract/Free Full Text]
  10. Corcoran TB, O'Shea A, Engel A, Shorten GD. The influence of propofol on P-selectin expression and nitric oxide production in re-oxygenated human umbilical vein endothelial cells. Acta Anaesthesiol Scand 50: 348–354, 2006.[CrossRef][Web of Science][Medline]
  11. Coulet F, Nadaud S, Agrapart M, Soubrier F. Identification of hypoxia-response element in the human endothelial nitric-oxide synthase gene promoter. J Biol Chem 278: 46230–46240, 2003.[Abstract/Free Full Text]
  12. Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399: 601–605, 1999.[CrossRef][Medline]
  13. Earley S, Walker BR. Increased nitric oxide production following chronic hypoxia contributes to attenuated systemic vasoconstriction. Am J Physiol Heart Circ Physiol 284: H1655–H1661, 2003.[Abstract/Free Full Text]
  14. Ehrenreich H, Rieckmann P, Sinowatz F, Weih KA, Arthur LO, Goebel FD, Burd PR, Coligan JE, Clouse KA. Potent stimulation of monocytic endothelin-1 production by HIV-1 glycoprotein 120. J Immunol 150: 4601–4609, 1993.[Abstract]
  15. Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 284: R1–R12, 2003.[Abstract/Free Full Text]
  16. Flusberg DA, Numaguchi Y, Ingber DE. Cooperative control of Akt phosphorylation, bcl-2 expression, and apoptosis by cytoskeletal microfilaments and microtubules in capillary endothelial cells. Mol Biol Cell 12: 3087–3094, 2001.[Abstract/Free Full Text]
  17. Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399: 597–601, 1999.[CrossRef][Medline]
  18. Gold ME, Bush PA, Ignarro LJ. Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun 164: 714–721, 1989.[CrossRef][Web of Science][Medline]
  19. Gooch KJ, Dangler CA, Frangos JA. Exogenous, basal, and flow-induced nitric oxide production and endothelial cell proliferation. J Cell Physiol 171: 252–258, 1997.[CrossRef][Web of Science][Medline]
  20. Graven KK, Farber HW. Endothelial hypoxic stress proteins. Kidney Int 51: 426–437, 1997.[Web of Science][Medline]
  21. Hampl V, Cornfield DN, Cowan NJ, Archer SL. Hypoxia potentiates nitric oxide synthesis and transiently increases cytosolic calcium levels in pulmonary artery endothelial cells. Eur Respir J 8: 515–522, 1995.[Abstract]
  22. Humbert M, Sitbon O, Simonneau G. Treatment of pulmonary arterial hypertension. N Engl J Med 351: 1425–1436, 2004.[Free Full Text]
  23. Hyoda K, Hosoi T, Horie N, Okuma Y, Ozawa K, Nomura Y. PI3K-Akt inactivation induced CHOP expression in endoplasmic reticulum-stressed cells. Biochem Biophys Res Commun 340: 286–290, 2006.[Web of Science][Medline]
  24. John TA, Ibe BO, Usha Raj J. Oxygen alters caveolin-1 and nitric oxide synthase-3 functions in ovine fetal and neonatal lung microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 291: L1079–L1093, 2006.[Abstract/Free Full Text]
  25. Justice JM, Tanner MA, Myers PR. Endothelial cell regulation of nitric oxide production during hypoxia in coronary microvessels and epicardial arteries. J Cell Physiol 182: 359–365, 2000.[CrossRef][Web of Science][Medline]
  26. Kondrikov D, Han HR, Block ER, Su Y. Growth and density-dependent regulation of NO synthase by the actin cytoskeleton in pulmonary artery endothelial cells. Am J Physiol Lung Cell Mol Physiol 290: L41–L50, 2006.[Abstract/Free Full Text]
  27. Lee YJ, Froelich CJ, Fujita N, Tsuruo T, Kim JH. Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and Akt cleavage. Clin Cancer Res 10: 1894–1900, 2004.[Abstract/Free Full Text]
  28. Liao JK, Zulueta JJ, Yu FS, Peng HB, Cote CG, Hassoun PM. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest 96: 2661–2666, 1995.[Web of Science][Medline]
  29. Luo WB, Wang YP. Magnesium lithospermate B inhibits hypoxia-induced calcium influx and nitric oxide release in endothelial cells. Acta Pharmacol Sin 22: 1135–1142, 2001.[Web of Science][Medline]
  30. Martinez AC, Stankevicius E, Jakobsen P, Simonsen U. Blunted non-nitric oxide vasodilatory neurotransmission in penile arteries from renal hypertensive rats. Vasc Pharmacol 44: 354–362, 2006.[CrossRef]
  31. Mateev S, Sillau AH, Mouser R, McCullough RE, White MM, Young DA, Moore LG. Chronic hypoxia opposes pregnancy-induced increase in uterine artery vasodilator response to flow. Am J Physiol Heart Circ Physiol 284: H820–H829, 2003.[Abstract/Free Full Text]
  32. McQuillan LP, Leung GK, Marsden PA, Kostyk SK, Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol Heart Circ Physiol 267: H1921–H1927, 1994.[Abstract/Free Full Text]
  33. Mies F, Shlyonsky V, Goolaerts A, Sariban-Sohraby S. Modulation of epithelial Na+ channel activity by long-chain n-3 fatty acids. Am J Physiol Renal Physiol 287: F850–F855, 2004.[Abstract/Free Full Text]
  34. Mildenberger E, Siegel G, Versmold HT. Locally released norepinephrine in the oxygen-dependent regulation of vascular tone of human umbilical vein. Pediatr Res 55: 267–272, 2004.[CrossRef][Web of Science][Medline]
  35. Murata T, Sato K, Hori M, Ozaki H, Karaki H. Decreased endothelial nitric-oxide synthase (eNOS) activity resulting from abnormal interaction between eNOS and its regulatory proteins in hypoxia-induced pulmonary hypertension. J Biol Chem 277: 44085–44092, 2002.[Abstract/Free Full Text]
  36. Murata T, Yamawaki H, Hori M, Sato K, Ozaki H, Karaki H. Hypoxia impairs endothelium-dependent relaxation in organ cultured pulmonary artery. Eur J Pharmacol 421: 45–53, 2001.[CrossRef][Web of Science][Medline]
  37. Palmer SK, Moore LG, Young D, Cregger B, Berman JC, Zamudio S. Altered blood pressure course during normal pregnancy and increased preeclampsia at high altitude (3100 meters) in Colorado. Am J Obstet Gynecol 180: 1161–1168, 1999.[CrossRef][Web of Science][Medline]
  38. Pearlstein DP, Ali MH, Mungai PT, Hynes KL, Gewertz BL, Schumacker PT. Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia. Arterioscler Thromb Vasc Biol 22: 566–573, 2002.[Abstract/Free Full Text]
  39. Phelan MW, Faller DV. Hypoxia decreases constitutive nitric oxide synthase transcript and protein in cultured endothelial cells. J Cell Physiol 167: 469–476, 1996.[CrossRef][Web of Science][Medline]
  40. Rasmussen LM, Hansen PR, Nabipour MT, Olesen P, Kristiansen MT, Ledet T. Diverse effects of inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase on the expression of VCAM-1 and E-selectin in endothelial cells. Biochem J 360: 363–370, 2001.[CrossRef][Web of Science][Medline]
  41. Searles CD. The nitric oxide pathway and oxidative stress in heart failure. Congest Heart Fail 8: 142–147, 2002.[Medline]
  42. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88: 1474–1480, 2000.[Abstract/Free Full Text]
  43. Simonsen U, Wadsworth RM, Buus NH, Mulvany MJ. In vitro simultaneous measurements of relaxation and nitric oxide concentration in rat superior mesenteric artery. J Physiol 516: 271–282, 1999.[Abstract/Free Full Text]
  44. Sohn HY, Krotz F, Gloe T, Keller M, Theisen K, Klauss V, Pohl U. Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. Cardiovasc Res 58: 638–646, 2003.[Abstract/Free Full Text]
  45. Stangl V, Lorenz M, Meiners S, Ludwig A, Bartsch C, Moobed M, Vietzke A, Kinkel HT, Baumann G, Stangl K. Long-term up-regulation of eNOS and improvement of endothelial function by inhibition of the ubiquitin-proteasome pathway. FASEB J 18: 272–279, 2004.[Abstract/Free Full Text]
  46. Stuehr DJ, Santolini J, Wang ZQ, Wei CC, Adak S. Update on mechanism and catalytic regulation in the NO synthases. J Biol Chem 279: 36167–36170, 2004.[Free Full Text]
  47. Su Y, Edwards-Bennett S, Bubb MR, Block ER. Regulation of endothelial nitric oxide synthase by the actin cytoskeleton. Am J Physiol Cell Physiol 284: C1542–C1549, 2003.[Abstract/Free Full Text]
  48. Su Y, Kondrikov D, Block ER. Cytoskeletal regulation of nitric oxide synthase. Cell Biochem Biophys 43: 439–449, 2005.[CrossRef][Web of Science][Medline]
  49. Tai SC, Robb GB, Marsden PA. Endothelial nitric oxide synthase: a new paradigm for gene regulation in the injured blood vessel. Arterioscler Thromb Vasc Biol 24: 405–412, 2004.[Abstract/Free Full Text]
  50. Takemoto M, Sun J, Hiroki J, Shimokawa H, Liao JK. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase. Circulation 106: 57–62, 2002.[Abstract/Free Full Text]
  51. Thomas SR, Chen K, Keaney JF Jr. Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway. J Biol Chem 277: 6017–6024, 2002.[Abstract/Free Full Text]
  52. Thompson LP, Aguan K, Pinkas G, Weiner CP. Chronic hypoxia increases the NO contribution of acetylcholine vasodilation of the fetal guinea pig heart. Am J Physiol Regul Integr Comp Physiol 279: R1813–R1820, 2000.[Abstract/Free Full Text]
  53. Toporsian M, Govindaraju K, Nagi M, Eidelman D, Thibault G, Ward ME. Downregulation of endothelial nitric oxide synthase in rat aorta after prolonged hypoxia in vivo. Circ Res 86: 671–675, 2000.[Abstract/Free Full Text]
  54. Villa LM, Salas E, Darley-Usmar VM, Radomski MW, Moncada S. Peroxynitrite induces both vasodilatation and impaired vascular relaxation in the isolated perfused rat heart. Proc Natl Acad Sci USA 91: 12383–12387, 1994.[Abstract/Free Full Text]
  55. Wadsworth R, Stankevicius E, Simonsen U. Physiologically relevant measurements of nitric oxide in cardiovascular research using electrochemical microsensors. J Vasc Res 43: 70–85, 2006.[CrossRef][Web of Science][Medline]
  56. White MM, McCullough RE, Dyckes R, Robertson AD, Moore LG. Chronic hypoxia, pregnancy, and endothelium-mediated relaxation in guinea pig uterine and thoracic arteries. Am J Physiol Heart Circ Physiol 278: H2069–H2075, 2000.[Abstract/Free Full Text]
  57. Whorton AR, Simonds DB, Piantadosi CA. Regulation of nitric oxide synthesis by oxygen in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 272: L1161–L1166, 1997.[Abstract/Free Full Text]
  58. Xiao D, Bird IM, Magness RR, Longo LD, Zhang L. Upregulation of eNOS in pregnant ovine uterine arteries by chronic hypoxia. Am J Physiol Heart Circ Physiol 280: H812–H820, 2001.[Abstract/Free Full Text]
  59. Xu XP, Pollock JS, Tanner MA, Myers PR. Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells. Cardiovasc Res 30: 841–847, 1995.[CrossRef][Web of Science][Medline]
  60. Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem 278: 15911–15916, 2003.[Abstract/Free Full Text]
  61. Zharikov SI, Block ER. Association of L-arginine transporters with fodrin: implications for hypoxic inhibition of arginine uptake. Am J Physiol Lung Cell Mol Physiol 278: L111–L117, 2000.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. A. Plock, N. Rafatmehr, D. Sinovcic, J. Schnider, H. Sakai, E. Tsuchida, A. Banic, and D. Erni
Hemoglobin vesicles improve wound healing and tissue survival in critically ischemic skin in mice
Am J Physiol Heart Circ Physiol, September 1, 2009; 297(3): H905 - H910.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
M. R. Andersen, U. Simonsen, N. Uldbjerg, C. Aalkjaer, and S. Stender
Smoking Cessation Early in Pregnancy and Birth Weight, Length, Head Circumference, and Endothelial Nitric Oxide Synthase Activity in Umbilical and Chorionic Vessels: An Observational Study of Healthy Singleton Pregnancies
Circulation, February 17, 2009; 119(6): 857 - 864.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/H2894    most recent
01230.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Østergaard, L.
Right arrow Articles by Simonsen, U.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Østergaard, L.
Right arrow Articles by Simonsen, U.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.