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Am J Physiol Lung Cell Mol Physiol 287: L525-L532, 2004. First published May 7, 2004; doi:10.1152/ajplung.00203.2003
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Greater vascularity, lowered HIF-1/DNA binding, and elevated GSH as markers of adaptation to in vivo chronic hypoxia

M. C. Tissot van Patot,1 J. Bendrick-Peart,1 V. E. Beckey,1 N. Serkova,1 and L. Zwerdlinger2

1Department of Anesthesiology, University of Colorado Health Sciences Center, Denver 80262; and 2St. Vincent’s General Hospital, Leadville, Colorado 80461

Submitted 24 June 2003 ; accepted in final form 3 May 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Vascularity is increased in placentas from high- compared with low-altitude pregnancies. An angiogenic response to hypoxia may protect an organ from further hypoxic insult by increasing blood flow and oxygen delivery to the tissue. We hypothesized that increased placental vascularity is sufficient to adapt to high altitude. Therefore, indexes of hypoxic stress would not be present in placentas from successful high-altitude pregnancies. Full-thickness placental biopsies were 1) collected and frozen in liquid nitrogen within 5 min of placental delivery and 2) fixed in formalin for stereologic analyses at high (3,100 m, n = 10) and low (1,600 m, n = 10) altitude. Hypoxia-inducible transcription factor (HIF-1) activity was analyzed by ELISA. Western blot analyses were used to evaluate HIF-1{alpha}, HIF-1{beta}, HIF-2{alpha}, von Hippel-Lindau protein, VEGF, Flt-1, enolase, and GAPDH. Magnetic resonance spectroscopy was used to evaluate endogenous metabolism. The ratio of placental capillary surface density to villous surface density was 70% greater at high compared with low altitude. HIF-1 activity and HIF-1-associated proteins were unchanged in placentas from high- vs. low-altitude pregnancies. Placental expression of HIF-1-mediated proteins VEGF, Flt-1, enolase, and GAPDH were unchanged at high vs. low altitude. Succinate, GSH, phosphomonoesters, and ADP were elevated in placenta from high compared with low altitude. Placentas from uncomplicated high-altitude pregnancies have greater vascularity and no indication of significant hypoxic stress at term compared with placentas from low altitude.

glutathione-SH; succinate; placenta; stereology

PREVIOUSLY, WE AND OTHERS have reported increased vascularity in placentas that have developed during healthy pregnancies at high compared with low altitude (3, 17, 30). A hypoxia-induced increase in vascularity is believed to assist in "rescuing" the tissue from hypoxia by increasing blood and thereby oxygen delivery to the area. Hypoxia can activate hypoxia-inducible transcription factor (HIF-1) (6, 7). HIF-1 enhances transcription of genes encoding hypoxia-sensitive proteins that are instrumental in protecting the tissue from hypoxia, such as erythropoietin, which increases the oxygen carrying capacity of the blood, VEGF and its receptor Flt-1, which stimulate angiogenesis, and glycolytic proteins such as GAPDH and enolase, which increase energy production through anaerobic glycolysis (8, 16, 25, 31).

If the response to hypoxia is successful, for example, vascularity (oxygen delivery) is increased sufficiently to protect the tissue from hypoxia, and the principle of negative feedback dictates that HIF-1 activity, expression of hypoxia-sensitive proteins, oxidative stress, and increased glycolysis should be attenuated. Because placental vascularity is established in early pregnancy, a hypoxia- or altitude-induced increase in vascularity probably occurs in early pregnancy as well (1). Therefore, establishing an increase in oxygen delivery early in gestation should protect the placentas from hypoxia so that term placentas of successful high-altitude pregnancies should not be associated with markers of hypoxic stress.

Thus we hypothesized that greater vascularity in placentas from uncomplicated pregnancies at high altitude would not be associated with markers of hypoxic metabolic stress including enhanced HIF/DNA binding, expression of HIF-related and hypoxia-sensitive proteins, and metabolic hypoxic markers. Our approach was to examine placentas from high- and low-altitude pregnancies to determine vascular responses, including capillary and villous surface densities; HIF/DNA binding activity; the presence of proteins associated with HIF-1 activation, including HIF-1{alpha}, HIF-2{alpha}, HIF-1{beta}, and von Hippel-Lindau protein (pvHL); and the expression of HIF-mediated proteins VEGF and Flt-1. We also determined metabolic stress markers, including GAPDH, enolase, and metabolic adaptation at each altitude. This strategy was designed to determine whether enhanced placental vascularity in hypoxic placentas is associated with indicators of hypoxic stress.

This study is important because hypoxia is implicated in the pathogenesis of many pregnancy complications, including preeclampsia, intrauterine growth restriction, and anemia, and poses a serious threat to the health of both fetus and mother (14). Fetal growth and development are impaired in the presence of hypoxia. However, successful pregnancy at high altitude represents successful adaptation to hypoxia. Therefore, determining the mechanisms of successful adaptation to chronic hypoxia during pregnancy and markers of adaptation failure may be a critical first step in determining successful therapeutic intervention in hypoxia-mediated diseases of pregnancy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Study design. Approval from the Colorado Multiple Institutional Review Board (COMIRB) at the University of Colorado Health Sciences Center was obtained to collect term placentas from uncomplicated, singleton gestational women at University Hospital in Denver, CO (1,600 m). Permission was also obtained from St. Vincent’s Hospital in Leadville, CO (3,100 m). Ten subjects at low altitude and ten subjects at high altitude consented according to COMIRB guidelines, and placentas were collected immediately following placental delivery. The subjects were between the ages of 18 and 34 yr, and gestational age was 39–41 wk. Two grams of tissue (full thickness of the placentas, including basal and chorionic plates) were collected from random locations and placed in liquid nitrogen within 5 min of placental delivery to stop the metabolic activity. Previous data from our laboratory (26) indicate that placental samples must be placed into liquid nitrogen within 9 min of placental delivery to avoid introducing artifacts by hypoxic/ischemic induction of glycolysis. The placenta was then dissected into five sections from which two blocks extending from basal plate to chorionic plate were dissected, placed into 10% formalin for 4 days, and then paraffin embedded at <58°C for stereologic analyses.

Immunhistochemistry. Placental sections were stained to label endothelium with a polyclonal mouse anti-human CD34+ antibody (1:20; BioGenex, Napa, CA), followed by an avidin-biotin complex reagent (Vectastain Elite; Vector Labs, Burlingame, CA) labeled with peroxidase for which 3',3'-diaminobenzidine (Sigma, St. Louis, MO) was used as a substrate. Negative controls were performed with mouse IgG in place of primary antibody.

Stereological analysis. Stereology was performed as previously reported (30). Briefly, four slides from four sections (4 µm) of each placenta and 16 fields per slide were evaluated. A 25-box microscope grid (25 x 25 µm) was used under x40 magnification; box size was chosen to minimize the number of capillaries per box. The guidelines of a 125-µm2 (25 µm2 per box) grid were applied, and capillary surface density of capillary luminal margins (Svcap) was calculated by 2 x total capillary intersects/number of points on villous tissue x total test line length [= 2D, where D = distance between points on grid (25 µm)] (3). We calculated the capillary (and villous) intersects by counting the villous tissue each time it crossed a horizontal line within the square lattice parameters specified (3). We calculated villous surface density (Svvill) in a similar manner, using the villous intersects as the numerator. A ratio of Svcap/Svvill is reported.

Western blot analysis. For immunoblot assays, 30 µg of total protein/lane or 50 µg of nuclear protein/lane were fractionated by electrophoresis with a NUPAGE 4–12% bis-Tris gradient gel (Invitrogen, Carlsbad, CA). The proteins were then transferred to a methanol-soaked polyvinylidene difluoride membrane by the semidry immunoblot method (Owl Model HEP-1 Panther Semi-Dry Electroblotter; Nunc, Rochester, NY). The membranes were immunoblotted with HIF-1{alpha}, HIF-1{beta}, HIF-2{alpha} (NB100–105, 100–124, 100–132, nuclear proteins; Novus Biologicals, Littleton, CO), pvHL, VEGF, or Flt-1 (Fl-181, C-1, H-225; Santa Cruz Biotechnology, Santa Cruz, CA); secondary antibodies conjugated with horseradish peroxidase (IgG-HRP) were used for detection and visualization by Pierce-SuperSignal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL). Images were visualized with the UVP BioChemi Imaging System, and relative quantification by densitometry was performed with LabWorks 4.0 software (UVP, Upland, CA). {beta}-Actin (A-5441, Sigma) was used as an internal control for protein loading, and data are expressed as a ratio of the protein of interest to {beta}-actin.

Enzyme-linked immunoabsorbance assay. An ELISA kit was used to assess HIF-1 activity on all samples in a single experiment (K2077-1; BD Biosciences, Clontech, Palo Alto, CA). Briefly, nuclear extract (20 µg) was added to a 96-well plate coated with the DNA consensus binding sequence for HIF-1. Bound HIF-1 was detected by the addition of mouse monoclonal primary antibody to HIF-1{alpha}, followed by HRP-conjugated secondary antibody. A microtiter plate reader (Multiskan Ascent; ThermoLab Systems, Helsinki, Finland) was used to measure the enzymatic product. An HIF-1 wild-type competitor oligonucleotide control was used to demonstrate DNA/HIF-1 binding specificity. Samples were run in duplicate, and coefficient of variation values were <10%.

Dual perchloric acid lipid extraction of placental tissues. To perform high-resolution magnetic resonance spectroscopy (MRS) on placental tissues, we extracted the frozen placental samples by a dual perchloric acid (PCA)/lipid extraction procedure developed in our laboratory (27). Snap-frozen tissues were powdered in a mortar grinder in the presence of liquid nitrogen. The powdered frozen tissue was added to 6 ml of ice-cold 12% PCA and subsequently homogenized with an electrical homogenizer Poly Tron PT 2100 (Kinematica, Lucerne, Switzerland). The PCA homogenates were put into an ice-cold ultrasound bath for 5 min. Then, the homogenates were centrifuged at 3,000 g and 4°C for 20 min. The aqueous phase was collected, and the pellet was resuspended with 2 ml of ice-cold PCA. The resuspended homogenates were put in an ultrasound bath and centrifuged again in the same conditions. The aqueous phase was added to the previously collected supernatant. The supernatants, containing placental water-soluble metabolites, were then neutralized with KOH, centrifuged for 20 min at 3,000 g and 4°C to remove potassium perchlorate, and lyophilized overnight for PCA extracts. The tissue pellets, remaining after the first centrifugations, were redissolved in 4 ml of ice-cold water. The redissolved pellets, containing placental lipids, were neutralized with KOH and lyophilized overnight for lipid extracts. The lyophilized PCA extracts, containing water-soluble metabolites, were reconstituted in 0.45 ml of deuterium oxide (Cambridge Isotope Laboratories, Andover, MA). The lyophilized lipid extracts were reconstituted in 1.5 ml of deuterated chloroform-methanol mixture (CDCl3/CD3OD, 2:1 vol/vol). After centrifugation, the supernatants were analyzed by MRS.

MRS on PCA and lipid extracts. To calculate the absolute concentrations of water-soluble and lipid metabolites, we carried out one-dimensional MRS experiments using a 500-MHz Bruker nuclear magnetic resonance (NMR) spectrometer with an Avance console (Bruker, Karlsruhe, Germany). A dual QNP 5-mm Bruker probe head was used for all experiments. For proton MRS, the operating frequency was 500 MHz, and a standard presaturation pulse program was used for water suppression. The other parameters were: 40 accumulations; 90° pulse angle; 0 dB power level; 7.35 µs pulse width; 10 parts per million (ppm) spectral width; and 12.85 s repetition time. Trimethylsilyl propionic-2,2,3,3,-d4 acid (TMSP, 0.6 mmol/l for PCA extracts and 1.2 mmol/l for lipid extract) was used as an external standard for the quantification of metabolites based on 1H-MRS signals. 1H chemical shifts were referenced to TMSP at 0 ppm. Before the 31P-MRS experiments were recorded, 100 mmol/l EDTA was added to each PCA extract for complexation of divalent ions. This resulted in 31P peaks with significant narrow line width (especially important for ATP signals). The pH was adjusted again to 7. The following NMR parameters with a composite pulse decoupling program were used: 202.1 MHz 31P operating frequency; 800 accumulations; 90° pulse angle; 12 dB power level for 31P channel; 9-µs pulse width; 35 ppm spectral width; and 2.0 s repetition time. The absolute concentration of glycerophosphocholine, calculated from 1H-MRS of the same extract, was used as an internal standard for quantification of phosphorus metabolites in 31P-MR spectra. The chemical shifts of {alpha}-ATP at –10 ppm were used as shift references. All MRS data were processed with the 1D WINNMR program (Bruker).

Statistical analyses. Stereology, densitometry, and ELISA data were analyzed by Student’s t-test. MRS data were analyzed by ANOVA. Scheffé’s post hoc test to was used to determine differences between variables. Significance for all statistical analyses was accepted at P ≤ 0.05. Data are presented as representative immunoblots indicating subject number in each lane and accompanied by densitometry analysis of immunoblots for the entire study group (n = 10 per altitude).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The characteristics of the subjects at 1,600 and 3,100 m were similar with respect to maternal age, gestational age of delivery, birth weight, placental weight, placental volume, and the ratio of placental weight to birth weight (Table 1).


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  		Table 1. Subject Characteristics

 
Placental stereologic analyses indicated that the ratio of capillary surface density (Svcap) to Svvill was greater in high- vs. low-altitude pregnancies (low altitude 5.65, high altitude 9.52, P = 0.03) (Ref. 30 and Fig. 1D). To determine whether greater vascularity protected the placentas from the hypoxic stress of high altitude, we determined HIF-1/DNA binding activity in placentas collected at high and low altitude. ELISA results indicated that HIF-1/DNA binding was 1.455 ng/ml (±0.182) at low and 0.476 ng/ml (±0.347) at high altitude, threefold less at high altitude (Fig. 2) (P = 0.0001).



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  		Fig. 1. Placental tissues from low- and high-altitude placentas were fixed in 10% buffered formalin, paraffin embedded, and immunohistochemically studied using anti-CD34+ antibody and hematoxylin background stain (x200). In all figures, low altitude is defined as 1,600 m and high altitude as 3,100 m. A: low-altitude placenta negative, using mouse IgG in place of primary antibody. B: low-altitude placenta (serial section from A). C: high-altitude placenta. D: ratio of capillary density to villous surface density (Svvill) in placentas from pregnancies at low (n = 10) and high (n = 10) altitude. EC, endothelial cells; V, villi; Svcap, capillary surface density. *P = 0.038.

 


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  		Fig. 2. Nuclear protein was extracted from low- and high-altitude placentas and analyzed for hypoxia-inducible transcription factor (HIF-1)/DNA binding by an ELISA transcription factor activity assay (n = 10 low-altitude and n = 10 high-altitude subjects). *P < 0.0001. Data are presented as means ± SE.

 
Because HIF-1 activity is primarily determined by the presence of the activated HIF-1{alpha} subunit in the nucleus, we determined the presence of nuclear HIF-1{alpha} by Western blot analyses. Nuclear HIF-1{alpha} was equivalent in placentas from low (n = 10)- and high (n = 10)-altitude pregnancies, as determined by densitometric analyses (Fig. 3). To further investigate reasons for less HIF-1 activity at high altitude, we determined nuclear HIF-1{beta}, to which HIF-1{alpha} must bind to create active HIF-1. Nuclear HIF-1{beta} was greater in placentas from high (n = 10)- compared with low (n = 10)-altitude pregnancies, as determined by densitometric analyses (Fig. 4). However, individual densitometry data indicated that placenta #3 expressed two- to threefold more HIF-1{beta} than other placentas (Fig. 4). When data were compared without placenta #3, there was no significant difference in HIF-1{beta} expression between low- and high-altitude placentas.



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  		Fig. 3. Top: nuclear proteins, extracted from low- and high-altitude placentas, were analyzed for HIF-1{alpha} and {beta}-actin by Western blot analysis. Bottom: data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (means ± SE).

 


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  		Fig. 4. Nuclear protein, extracted from placentas from low- and high-altitude pregnancies, were analyzed for HIF-1{beta} and {beta}-actin by Western blot analysis (top). Data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (bottom). The graph on the left depicts individual data to demonstrate the variability of HIF-1{beta} between subjects, whereas the graph on the right depicts the mean ± SE at each altitude with and without #3 at high altitude. *P = 0.033 greater than low altitude.

 
The pvHL binds to HIF-1{alpha} during normoxia, targeting HIF-1{alpha} for ubiquitination and proteosomal degradation. In the presence of normoxia, as suggested by unaltered HIF-1{alpha} at high altitude, there should be no difference in pvHL at high compared with low altitude. In Western blot analysis of total placental protein extracts from 10 low- and 10 high-altitude pregnancies, there were, as expected, no differences in pvHL expression (Fig. 5). Thus less HIF-1 activity in placentas from high- vs. low-altitude pregnancies was not associated with any change in the presence of HIF-1{alpha} or -1{beta} subunits or expression of pvHL.



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  		Fig. 5. Top: total protein, extracted from low- and high-altitude placentas, was analyzed for von Hippel-Lindau (vHL) protein and {beta}-actin by Western blot analysis. Bottom: data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (means ± SE).

 
In some tissues, HIF-2 is activated under less severe hypoxic conditions than HIF-1 and may be responsible for increased transcription of hypoxia-sensitive genes (32). To determine whether placental tissue at high altitude was responding to hypoxia via HIF-2 rather than HIF-1, we analyzed placentas from 10 low- and 10 high-altitude pregnancies for nuclear HIF-2{alpha} by Western blot analyses (Fig. 6). HIF-2{alpha} was not different between placental nuclear extracts from low- and high-altitude pregnancies, although there was a trend toward greater expression at high altitude, which may prove significant in a larger sample of placentas.



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  		Fig. 6. Top: nuclear protein, extracted from placentas from low- and high-altitude pregnancies were analyzed for HIF-2{alpha} and {beta}-actin by Western blot analysis. Bottom: data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (means ± SE).

 
To further test whether placentas at high altitude are experiencing hypoxic stress, we analyzed expression of hypoxia-sensitive angiogenic and glycolytic proteins. Expression of VEGF, a hypoxia-sensitive angiogenic protein, was not altered in placentas from high (n = 10) compared with low (n = 10) altitude as determined by Western blot and densitometric analysis (Fig. 7).



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  		Fig. 7. Top: total protein, extracted from low- and high-altitude placentas, was analyzed for VEGF, Flt-1, and {beta}-actin by Western blot analysis. Bottom: data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (means ± SE).

 
Because a hypoxia-stimulated increase in the VEGF receptor Flt-1 could increase the biological activity of VEGF, further Western blot analyses for Flt-1 expression in 10 low- and 10 high-altitude placentas were performed, and densitometric analysis indicated that expression did not differ between placentas from high- vs. low-altitude pregnancies (Fig. 7).

Because cellular response to hypoxia initiates an increase in expression of glycolytic enzymes and hence glycolysis, we determined the expression of glycolytic enzymes enolase and GAPDH placentas from low (n = 10)- and high (n = 10)-altitude pregnancies by Western blot and subsequent densitometric analyses. There was no change in the expression of placental enolase and GAPDH between low- and high-altitude pregnancies (Fig. 8).



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  		Fig. 8. Top: total protein, extracted from low- and high-altitude placentas, was analyzed for GAPDH, enolase, and {beta}-actin by Western blot analysis. Bottom: data are expressed as the ratio of relative densitometry units of the protein of interest to {beta}-actin for total protein (means ± SE).

 
Because there was no evidence of HIF-1 activation or glycolytic stress, we sought to determine, utilizing MRS, whether there were metabolic markers for hypoxia at high compared with low altitude. 1H and 31P-MRS analysis indicated that metabolites succinate, glutathione-SH (GSH), phosphomonoesters (PME), and ADP were increased in placentas collected at high altitude (Table 2). Thus the ratio of PME to phosphodiesters was also increased; however, the ATP/ADP ratio did not change, since ATP also showed a tendency to increase. No significant increase in lactate, a marker for anaerobic glycolysis, was seen. There were no changes in polyunsaturated fatty acid (PUFA) concentrations in lipid spectra of high- vs. low-altitude placentas, indicating no increase in lipid peroxidation (Table 3).


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  		Table 2. Metabolic markers at high and low altitude

 

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  		Table 3. Lipid spectra in low- and high-altitude placentas

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, the main finding was that placentas from successful pregnancies exposed to hypoxia throughout gestation had greater placental vascularity and no evidence of severe hypoxic stress. Placental HIF-1/DNA binding activity was actually lower at altitude, and there was no increase in the expression of hypoxia-sensitive proteins. Interestingly, the reduction in HIF-1 activity was not associated with changes in pvHL, nuclear HIF-1{alpha}, or HIF-1{beta}, nor was HIF-2{alpha} increased in the nucleus. In regard to metabolism, succinate was elevated as is often found during hypoxia; however, GSH was also elevated. Furthermore, there was no evidence of lipid peroxidation or glycolytic activity.

The reduction in HIF-1 activity was not caused by introducing hypoxia during placental collection, as all placental samples were minced and collected into liquid nitrogen within 5 min of vaginal delivery and stored at –80°C until nuclear proteins were extracted for analyses. Furthermore, MRS analysis did not indicate acute hypoxic insult in any of the tissues, since no increase in lactate nor decrease in the ATP/ADP ratio or glucose was seen. Each Western blot analysis was performed a minimum of three times, and each ELISA sample was analyzed in triplicate (coefficient of variance <10%), producing consistent results each time. The number of subjects within each group (n = 10 low-, n = 10 high-altitude placentas) was sufficient to achieve 98% power, showing less HIF-1 activity at high altitude.

The investigators are aware that Denver is not at sea level. Low-altitude placentas in this study were collected at 1,600 m, where the partial pressure of inspired oxygen (PIO2) is 122 mmHg compared with 149 mmHg at sea level. Although the change in PIO2 at 1,600 m is not enough to cause altitude-induced illness (13), there may be metabolic, enzymatic, or protein changes in tissue without associated clinical complications. Therefore, future studies examining sea-level placental tissue compared with those at 1,600 and 3,100 m are planned.

Because these placentas were collected following labor and delivery, it is possible that changes in HIF-1 activity occurred as a result of labor and delivery and did not reflect in vivo values before labor. Ideally, placentas from Cesarean sections should be used to most closely assess in vivo values. This was a potential problem for our collections in Leadville, CO, as all preplanned Cesarean sections are performed in one of several low-altitude facilities, making Cesarean section material impractical for collection in our study design. However, in a large number of placentas from high-altitude pregnancies, all had less HIF-1 activity, suggesting that the results are reliable.

Because HIF-1 is important in promoting transcription of VEGF, Flt-1, GAPDH, and enolase and because HIF-1 activity was not greater at high compared with low altitude, it is not surprising that there was no increase in the expression of hypoxia-sensitive proteins. Although VEGF and Flt-1 are important for promoting vasculogenesis (2, 15, 21), it is not surprising that there was no increased expression in the highly vascular placentas. Placental vascular development occurs primarily during the first and early second trimesters and is most likely no longer taking place at term. Furthermore, failure to increase expression of glycolytic enzymes GAPDH and enolase at high altitude was supported by MRS data indicating no change in glucose, lactate, or ATP in high-altitude placentas. Although with the glycolytic enzymes, protein expression may not change, whereas the activity of the enzyme might, changes in molecules such as ADP suggest this may be the case.

Metabolic profiles of our high-altitude placental tissue did not reveal changes characteristic for acute short-term hypoxia (Table 3) (4, 22, 28, 29). This suggests that placentas from uncomplicated high-altitude pregnancies are not exposed to hypoxic conditions (possibly due to increased vascularity) but, rather, showed metabolic adaptation to increased oxygen delivery. Phosphomonoesters, precursors for membrane phospholipids, were increased, indicating increased membrane synthesis, in accordance with the data indicating greater capillary development in high-altitude placentas. ADP was elevated, but the ATP/ADP ratio indicating energy state was unchanged. ATP may be converted to ADP more quickly to provide energy for enhanced membrane synthesis.

Previous studies on hypoxic tissue indicate that succinate is increased during hypoxia, while GSH is reduced (4, 18, 28). During hypoxia, complex II in the mitochondrial respiratory chain appears to switch from succinate dehydrogenase to fumerate reductase, resulting in an accumulation of succinate (18). Because succinate is produced in the mitochondrial respiratory chain, succinate concentrations are dependent on tissue PO2 (18). GSH is reduced during hypoxia by conversion to glutathione-S-S-glutathione and is more dependent on oxygen content than PO2 (4). Previous studies in which succinate was greater and GSH lower during hypoxia were designed such that PO2 and oxygen content were reduced. In contrast, in the current study, placentas from high-altitude pregnancies most likely experienced lowered PO2 as a result of hypobaric hypoxia but greater oxygen content as a result of greater vascularity. Therefore, we propose that the greater succinate concentration was due to the lower PO2 and the greater GSH concentration was due to greater oxygen content. Also, the equal concentrations of PUFA in low- and high-altitude placentas indicated no evidence of increased lipid peroxides (30), related to hypoxia.

Because GSH has been reported to reduce HIF-1 activity when exogenously administered to hypoxic tissue (10, 11), greater GSH concentrations may have inhibited the activity of HIF-1 in our high- compared with low-altitude placentas. Our findings regarding HIF activity do not dispute what others have found: that the placentas can increase HIF activity when hypoxic and have greater HIF-1{alpha} early rather than late in pregnancy (5, 9, 23, 24). Canniggia et al. (5) reported that in normal, low-altitude pregnancies placental HIF was elevated in early gestation (before 10 wk) during the most severe placental hypoxia but decreased as the placenta invaded the uterus and was exposed to maternal circulation. The altitude-induced increase in placental vascularity probably occurred during the same early gestation time period; however, that could not be determined at this time, as sampling of early-pregnancy placentas was not possible in our study.

Data from the literature report HIF to be consistently elevated during hypoxia in pathological conditions (25). Similarly, our current study reports no increase in HIF activity in placentas from women who successfully completed pregnancy at high altitude. Therefore, we consider that lack of HIF activity in placentas collected at high altitude does not represent a pathologic condition but is rather a surrogate marker for successful adaptation to high altitude. Our data suggesting that reduced HIF-1 activity represents adaptation to high altitude are supported by a report from Hochachka and Rupert (12), in which Andean natives had lower erythropoietin synthesis in response to hypoxia than lowlanders; however, the genetic sequences encoding erythropoietin and HIF-1{alpha} were unchanged in the native population. Hochachka and Rupert hypothesized that their results suggested "that the altered erthropoietic response in Andean natives reflects adaptations in hypoxia sensing, rather than hypoxia response, mechanisms" (12).

For example, failure to attain normal pregnancy at high altitude most often results in preeclampsia at rates three to four times those of low-altitude pregnancies (19), and HIF is elevated 1.5- to 2.5-fold in placentas from preeclamptic pregnancies at low altitude (24). Preeclampsia is characterized by greatly elevated blood pressure, poor placental development, and impaired placental blood flow (20). In contrast, the current study indicates greater placental vascular development and less HIF activity in placentas from normal pregnancies at high altitude.

Long-term hypoxia is a complication of many diseases, including pregnancy-related disorders and pulmonary and cardiovascular diseases. Determining the mechanisms by which tissues successfully adapt to chronic hypoxia is crucial for survival of tissues challenged by chronic hypoxia. Our data suggest that greater GSH concentration and less HIF activity may be implicated in the mechanism of successful adaptation to chronic hypoxia. However, our study design did not allow for establishing cause-effect relationships. It remains to be evaluated whether the observed changes are evidence of adaptation or markers for a yet-unidentified mechanism.


   ACKNOWLEDGMENTS
 
The authors thank Drs. John Reeves and Uwe Christians for invaluable mentorship and assistance in preparing this manuscript. We also acknowledge the invaluable assistance of the nursing staff on the labor and delivery wards of University Hospital, Denver, and St. Vincent’s General Hospital, Leadville, CO, without whom this study would not have been feasible. Also, we thank St. Vincent’s General Hospital for providing excellent facilities and a supportive collaborative environment in which to work.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. C. Tissot van Patot, Dept. Anesthesiology, B-113, Univ. of Colorado Health Sciences Center, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: martha.tissotvanpatot{at}uchsc.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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