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. Author manuscript; available in PMC: 2020 Jun 1.
Published in final edited form as: Hypertension. 2019 Jun;73(6):1319–1326. doi: 10.1161/HYPERTENSIONAHA.119.12641

High Altitude Reduces NO-dependent Myometrial Artery Vasodilator Response During Pregnancy

Ramón A Lorca 1,*, Sydney L Lane 1, Elise S Bales 1, Hisham Nsier 3, HeaMi Yi 3, Meghan A Donnelly 2, Anna G Euser 2, Colleen G Julian 4, Lorna G Moore 1
PMCID: PMC6506366  NIHMSID: NIHMS1525783  PMID: 31006328

Abstract

The chronic hypoxia of high-altitude residence reduces uterine artery blood flow during pregnancy, likely contributing to an increased frequency of preeclampsia and intrauterine growth restriction. We hypothesized that this lesser pregnancy blood flow rise was due, in part, to reduced vasodilation of myometrial arteries. Here we assessed myometrial artery vasoreactivity in healthy residents of high (2902±39m) or low altitude (1669±10m). Myometrial artery contractile responses to potassium chloride, phenylephrine or the thromboxane A2 agonist U46619 did not differ between low- and high-altitude women. Acetylcholine vasodilated phenylephrine- or U466119 pre-constricted myometrial arteries at low altitude, yet had no effect on high-altitude myometrial arteries. In contrast, another vasodilator, bradykinin, relaxed myometrial arteries from both altitudes similarly. At low altitude, the nitric oxide synthase inhibitor L-NAME decreased both acetylcholine and bradykinin vasodilation by 56% and 33%, respectively. L-NAME plus the cyclooxygenase inhibitor indomethacin had similar effects on acetylcholine and bradykinin vasodilation (68% and 42% reduction, respectively) as did removing the endothelium (78% and 50% decrease, respectively), suggesting a predominantly nitric oxide-dependent vasodilation at low altitude. However, at high altitude, L-NAME did not change bradykinin vasodilation, whereas indomethacin or endothelium removal decreased it by 28% and 72%, respectively, indicating impaired nitric oxide signaling at high altitude. Suggesting that the impairment was downstream of endothelial nitric oxide synthase, high altitude attenuated the vasodilation elicited by the nitric oxide donor sodium nitroprusside. We concluded that reduced nitric oxide-dependent myometrial artery vasodilation likely contributes to diminished uteroplacental perfusion in high-altitude pregnancies.

Keywords: pregnancy, vasodilation, hypoxia, high altitude, myometrial artery

Summary

Residence at high altitude does not affect myometrial artery vasoconstrictor responses during pregnancy but decreases the vasodilatory responses to acetylcholine as the result of reduced NO-dependent signaling. The vasodilator response to bradykinin is unaffected but changed from being largely NO-dependent at LA to NO-independent at HA. Reduced NO signaling is likely a key mechanism by which chronic hypoxia reduces uterine vascular adaptation to pregnancy.

INTRODUCTION

The chronic hypoxia of high-altitude residence (HA, >2500 m) raises the incidence of preeclampsia and intrauterine growth restriction (IUGR) threefold.1–3 Contributing to these pregnancy disorders is a lesser rise in uterine artery blood flow across gestation, suggesting that chronic hypoxia impairs uterine vascular adaptation to pregnancy.4–8 While hypoxia is a consequence of uterine vascular impairment, the increased incidence of these pregnancy disorders at HA suggests that hypoxia contributes to their etiology as well.

Considerable experimental animal data indicate that chronic hypoxia interferes with the normal uterine vascular responses to pregnancy. The normal growth of the main uterine artery is impaired in chronically-hypoxic pregnant guinea pigs due, in part, to less pregnancy-associated rise in DNA synthesis in all layers of the uterine artery wall.9 Fourth-generation uterine vessels from sheep chronically exposed to high altitude have increased myogenic tone, less potassium channel-induced vasodilation10–13 and augmented oxidative stress.14 Pregnant rats and guinea pigs exposed to hypoxia during pregnancy also have diminished vasodilator responses15, 16 and less nitric oxide (NO)-dependent vasodilation.17

Impaired vasodilation of myometrial arteries (MA) and incomplete remodeling of uterine spiral arteries are likely the primary causes of the reduced uteroplacental perfusion characterizing IUGR and preeclampsia.4–6, 18 MA are key determinants of uterine vascular resistance and uteroplacental blood flow during human pregnancy given the pronounced increases in the diameters of both the upstream and downstream vessels.19 Demonstrating the importance of MA vascular responses, MA from preeclamptic pregnancies show reduced vasodilator responses to flow and vasodilating agents compared to those from uncomplicated pregnancies.4–6, 20 However, whether chronic hypoxia affects human MA vasoreactivity during normal pregnancy is unknown.

Here, we tested the hypothesis that residence at HA reduces human MA vasodilation compared to low altitude (LA, <1700 m) in uncomplicated pregnancies. In phenylephrine (PE) or U46619-pre-constricted vessels, we observed that the vasodilatory responses to acetylcholine (ACh) were blunted in MA from HA vs. LA. Although bradykinin (BK)-dependent vasodilation was similar at both altitudes, the NO contribution differed insofar as vasodilation was largely NO-dependent at LA but NO-independent at HA. These observations suggest that impaired NO-dependent vasodilation of the MA may be a key contributor to reduced uterine artery blood flow and fetal growth in HA pregnancies.

METHODS

All data supporting the findings of this study will be available on request from the authors. For detailed Methods see online-only Data Supplement.

Study subjects and tissue samples.

Subjects were classified in two groups: those living above 2500 m (HA, n=20 subjects) and those living below 1700 m (LA, n=40 subjects). Subject inclusion criteria were: maternal age from 18–45 years, pre-pregnant body mass index less than 30 kg/m2, we excluded subjects with known risk factors for IUGR (i.e., diabetes, chronic hypertension, smoking), or diagnoses during the current pregnancy of IUGR, preeclampsia or the presence of fetal congenital anomalies. Prior to sample collection, all subjects signed written consent forms approved by the University of Colorado Multiple Institutional Review Board (14–2178) or the Catholic Health Initiative Institute for Research and Innovation Institutional Review Board (1310). Myometrial biopsies were processed as detailed in online-only Data Supplement.

Myometrial vascularization measurements.

Myometrial samples (0.5 cm3 sections) were fixed in 4% paraformaldehyde (Affymetrix, Cleveland, OH), stained with anti-CD31 and α-actin antibodies. Number and size of vessels (as identified by CD31 staining) were analyzed using STEPanizer and Image J software, respectively.

MA vasoreactivity.

MA were cleaned from connective tissue in ice-cold phosphate saline buffer (PBS, Thermo Fisher Scientific, Waltham, MA) and mounted in a small-vessel wire myograph (Multi Wire Myograph System 620M, DMT-USA, Ann Arbor, MI) with two wires (40-μm diameter) threaded through the vessel lumen and connected to either a tension transducer or micrometer. The vessels were normalized to an internal diameter of 0.9 of L13.3kPa in a chamber containing oxygenated (95% O2/5% CO2) and warmed (37°C) Krebs buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, and 11 mM D-glucose). After at least 45 min of equilibration, MA were constricted with 60 mM KCl (Sigma-Aldrich, St. Louis, MO) to establish viability; vessels failing to constrict at least 1 mN (estimated as an appropriate signal-to-noise ratio for tension measurement) were excluded from further study. Vessels were pre-constricted with either 10 μM phenylephrine (PE, Sigma-Aldrich; 78.8±3.7% of maximal PE response at LA and 87.8±3.5% at HA) or 1 μM U46619 (Cayman Chemical, Ann Arbor, MI), a thromboxane A2 agonist (99.9±0.01% of maximal U46619 response at LA and 98.9±0.7% at HA), and the vasodilator response to either acetylcholine or bradykinin (ACh, 0.1 nM-100 μM; BK, 0.1 nM-1 μM; Thermo Fisher Scientific) was evaluated, with each dosage being applied for 3–5 min before achieving the next concentration.

To assess the contributions of endothelial factors, vessels were incubated for 20 min with either the nitric oxide (NO) synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME, 10 μM; BioVision Inc., Milpitas, CA) or L-NAME (10 μM) plus the cyclooxygenase (COX) inhibitor indomethacin (Indo, 10 μM; Sigma-Aldrich) before PE-constriction and the application of ACh or BK. In a subset of MA, the endothelial cell layer was mechanically disrupted by rolling a hair through the vessel lumen; loss of endothelial function was judged as < 30% relaxation to 10 μM ACh or 10 nM BK. To assess changes in NO signaling, HA or LA MA were pre-constricted with 10 μM PE and then vasodilated with the NO donor sodium nitroprusside (SNP, 0.1 nM-100 μM). ACh, BK and SNP relaxation are shown as percentage of PE or U46619 contraction. Area under the curve (AUC) was calculated using Graph Pad 7 software (San Diego, CA).

Western blot analysis.

Myometrial lysates were tested for total endothelial NO synthase (eNOS), Ser-1177 phosphorylated eNOS or CD31.

Statistical Analyses.

Sample sizes are shown as the number of subjects followed by the total number of vessels. In vascular function experiments, vessels from the same subject under the same conditions were averaged, in average 3.1±0.2 (ranging from 1 to 7) vessels per subject were used. The effects of drug treatment or altitude were determined using 2-way ANOVA followed by Sidak’s multiple comparisons or by non-parametric Mann-Whitney U test (Graph Pad 7 software) as needed. Demographic, immunohistochemistry and western blot data were analyzed by non-parametric Mann-Whitney U test or chi-square analysis (Graph Pad 7 software) as needed. A p-value < 0.05 was considered significant.

RESULTS

Subject characteristics and myometrial vascular parameters

Maternal age, height, pre-gravid body mass index, parity and ethnicity, as well as infant length at birth and sex were similar in the LA and HA pregnancies (Table 1). However, the birth weight of babies born to women living at HA were 11% lower than those born at LA (p<0.001, Table 1). In addition, ponderal index and, marginally, head circumference but not length were also reduced by altitude (p<0.001 and <0.05, respectively) consistent with lower birth weights being due to fetal growth restriction rather than constitutional factors (Table 1). Gestational age was slightly (but clinically insignificantly) shorter in HA vs. LA pregnancies (p<0.05, Table 1).

Table 1.

Maternal and infant characteristics.*

Characteristic LA
(n=40)† 		HA
(n=20)† 		p value
Maternal
Age at delivery (y) 32.9 ± 0.8 32.2 ± 1.4 0.3630
Height (cm) 164.2 ± 1.4 165.5 ± 1.1 0.3636
Pre-gravid BMI (kg/m2)‡ 25.3 ± 0.4 23.9 ± 0.8 0.1616
Parity (no. of live births) 2.2 ± 0.1 2.2 ± 0.2 >0.9999
Ethnicity (% of Hispanic) 15 35 0.0763
Altitude of residence (m) 1669 ± 10 2902 ± 39 <0.001
Infant
Gestational age (weeks) 39.2 ± 0.1 39.0 ± 0.1 0.0451
Birth weight (g) 3502 ± 72 3107 ± 79 <0.001
Length (cm) 50.1 ± 0.3 49.7 ± 0.5 0.3039
Head circumference (cm) 35.0 ± 0.2 34.3 ± 0.3 0.0372
Ponderal Index 2.78 ± 0.04 2.55 ± 0.06 <0.001
Sex (% female) 45 37 0.5535
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*

All values are means ± SEM.

†

n=number of subjects. p values were estimated by non-parametric Mann-Whitney U test or chi-square analysis.

‡

BMI, body mass index.

The numbers and sizes of myometrial blood vessels were similar in women living at LA and HA as demonstrated by the lack of differences in either the vascular volume fraction or average blood vessel perimeter (Figure 1).

Figure 1. Myometrial vascular volume fraction and vessel size are not affected by altitude.

Figure 1.

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Representative microscope pictures of myometrial tissue from pregnant women residing at LA (A) or HA (B) showing staining of endothelial cells (CD31, green) and smooth muscle cells (α-SMA, red). White arrows show blood vessel, scale bars=50 μm. C, volume fraction quantification (mean values, 0.13 ± 0.01 at LA and 0.12 ± 0.01 at HA, n=15 and 10 subjects, respectively). D, blood vessel perimeter quantification (mean values, 21.0 ± 2.3 μm at LA and 19.6 ± 2.9 μm at HA, n=15 and 10 subjects, respectively). Symbols are averaged values for each subject, bars are median values. Same italicized letters represent no statistical differences between LA and HA.

Vasoconstrictor responses to KCl, PE and U46619

MA from LA and HA vasoconstricted similarly to increasing concentrations of KCl as shown by the absence of differences in maximal force or EC50 whether the latter was expressed as absolute force or normalized to Kmax (Supplemental Figure S1, Table S1). Likewise, there were no altitudinal differences in the MA vasoconstrictor responses to PE or U46619 as measured by the maximal force or normalized to Kmax (Supplemental Figure S1, Table S1).

ACh vasodilator response in MA

In LA vessels, PE pre-constricted MA vasodilated in response to ACh in a concentration-dependent manner, but HA MA had a blunted vasodilator response to ACh (p<0.05, Figure 2A). Using a different vasoconstrictor, U46619, to pre-constrict MA, ACh also vasodilated LA MA to a greater extent than in HA MA (p<0.05, Figure 2B).

Figure 2. Vasodilation induced by ACh is reduced by HA in MA.

Figure 2.

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ACh concentration-response curves expressed as percentage of 10 μM PE (A, circles, n=9 subjects, 15 vessels at LA and 5 subjects, 12 vessels at HA) or 1 μM U46619 response (B, diamonds, n=6 subjects, 10 vessels at LA and 4 subjects, 6 vessels at HA) in MA from LA (closed symbols) or HA (open symbols). Before ACh curves were performed, MA from LA (C, closed symbols) or HA (D, open symbols) were pretreated with 10 μM L-NAME (squares, n=5 subjects, 9 vessels at LA and 5 subjects, 5 vessels at HA) or 10 μM L-NAME + 10 μM Indo (diamonds, n=4 subjects, 7 vessels at LA and 5 subjects, 5 vessels at HA), or their endothelium was removed (- endothelium, triangles, n=4 subjects, 5 vessels at LA and 6 subjects, 9 vessels at HA). E and F, AUC analysis of curves in panels C and D, respectively. Symbols in A-D are mean values, bars are SEM; symbols in E and F are averaged vessels per subject, lines are medians. Different italicized letters represent statistical differences with a p<0.05.

To evaluate the contribution of endothelial signaling, ACh-dependent vasodilation was compared in MA with and without prior incubation with L-NAME, L-NAME plus Indo, or endothelium removal in PE-pre-constricted vessels. Pre-incubation with L-NAME diminished ACh vasodilation in LA MA (p<0.05, Figure 2C and E) as did removal of the endothelial layer (p<0.05, Figure 2C and E). Incubation of LA MA with L-NAME plus Indo reduced ACh vasodilation to a similar extent compared to that observed with L-NAME alone (p<0.05, Figure 2C and E). At HA, there was no effect of either incubation with L-NAME alone, L-NAME plus Indo, or endothelial removal (Figure 2D and F) on ACh vasodilation.

To consider the possibility that ACh may be exerting a vasoconstrictor effect that contributed to the lack of vasodilation observed at HA, we tested the effect of ACh without pre-constricting the MA. ACh had no effect on HA MA whether administered alone, following incubation with L-NAME or L-NAME plus Indo, or in endothelium-denuded vessels (Supplemental Figure S2).

BK vasodilator response in MA

To determine whether the blunted vasodilator responses observed in HA MA was confined to ACh, we tested another vasodilator, BK. PE pre-constricted MA from both LA and HA vasodilated in response to BK (Figure 3A). BK-dependent vasodilation was also observed when MA were pre-constricted with U46619 at both LA and HA (Figure 3B).

Figure 3. Vasodilation induced by BK is not affected by HA in MA.

Figure 3.

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BK concentration-response curves expressed as percentage of 10 μM PE (A, circles, n=14 subjects, 27 vessels at LA and 6 subjects, 13 vessels at HA) or 1 μM U46619 response (B, diamonds, n=6 subjects, 11 vessels at LA and 5 subjects, 10 vessels at HA) in MA from LA (closed symbols) or HA (open symbols). Before BK curves were performed, MA from LA (C, closed symbols) or HA (D, open symbols) were pretreated with 10 μM L-NAME (squares, n=7 subjects, 15 vessels at LA and 5 subjects, 10 vessels at HA) or 10 μM L-NAME + 10 μM Indo (diamonds, n=5 subjects, 12 vessels at LA and 5 subjects, 6 vessels at HA), or their endothelium was removed (- endothelium, triangles, n=6 subjects, 9 vessels at LA and 5 subjects, 8 vessels at HA). E and F, AUC analysis of curves in panels C and D, respectively. Symbols in A-D are mean values, bars are SEM; symbols in E and F are averaged vessels per subject, lines are medians. Different italicized letters represent statistical differences with a p<0.05.

At LA, the MA vasodilator response to BK was partially blunted by pre-incubation with L-NAME, L-NAME plus Indo or by removing the endothelium (p<0.05, Figure 3C and E), with the effects of L-NAME and L-NAME plus Indo being indistinguishable (Figure 3C and E). Interestingly, although endothelial removal reduced the vasodilator response to BK at HA (p<0.05, Figure 3D and F), there was less dependence on NO-dependent vasodilation as indicated by the lack of an effect of L-NAME (Figure 3D and F) and only partial attenuation of BK vasodilation achieved by L-NAME + Indo (p<0.05, Figure 3D).

NO signaling

To address whether a reduction in NO signaling could explain the lack of effect of L-NAME on BK-dependent vasodilation of HA MA, we analyzed the activation of eNOS in myometrial samples from LA and HA women as measured by phosphorylation of residue Ser-1177. Total amounts of eNOS and Ser-1177 phosphorylated eNOS protein were similar in myometrial samples from HA and LA women (Figure 4A).

Figure 4. HA reduced NO-dependent vasodilation but not eNOS expression.

Figure 4.

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A, Western blot analysis of eNOS activation in myometrial tissue samples from LA and HA. i, immunoblot detection of eNOS, Ser-1177 phosphorylated eNOS (p-eNOS) and CD31 in myometrium from LA and HA. Negative (−) and positive (+) eNOS controls are mouse lung tissue lysates from eNOS knockout and wild type mice, respectively. ii-iv, densitometric quantification of eNOS, p-eNOS and the ratio p-eNOS/eNOS in human myometrium from LA (closed circles, n=4 subjects) or HA (open circles, n=4 subjects). B, SNP concentration-response curves expressed as percentage of 10 μM PE response in MA from LA (closed symbols, n=7 subjects, 18 vessels) or HA (open symbols, n=4 subjects, 10 vessels). C, AUC analysis of curves in panel B. Symbols in A are individual values, lines are medians; symbols in B are mean values, bars are SEM; symbols in B are averaged vessels per subject, lines are medians. Different italicized letters represent statistical differences with a p<0.05.

Since basal eNOS activity did not change between LA and HA, we assessed the role of downstream NO signaling pathways on ACh vasodilation by examining the effect of the NO donor SNP in the MA from LA and HA women. Even though SNP vasodilated the vessels from both altitudes, the response to SNP in the HA MA was attenuated compared with that seen in the LA MA (p<0.05, Figure 4B and C) indicating a likely impairment in downstream, soluble guanylate cyclase/protein kinase G (sGC/PKG) pathways.

DISCUSSION

Given the important role of the MA in the regulation of uterine vascular resistance19 and prior observations that uterine blood flow is reduced during high- compared with low-altitude pregnancy, we tested whether MA vasodilator function is impaired under conditions of HA. Our study results showed that residence at HA reduced ACh-dependent vasodilation in MA from healthy pregnant women due to impaired NO signaling. The lack of ACh vasodilation was not the result of altitudinal differences in vasoconstrictor responses since the responses to several agonists (KCl, PE, U46619) were identical, and therefore the effect of HA was specific to MA vasodilation. There were also no differences between altitudes in the number or size of the MA, possibly due to the fact that the myometrial samples used in this study were not obtained from the site of placentation and indicating that the effect of altitude was specific to vasodilator function of the MA themselves. The lack of MA vasodilation appeared to be the result of impaired NO signaling given that L-NAME had no effect on BK vasodilation in HA MA whereas MA vasodilation was largely dependent on increased NO production at LA. Thus, while HA MA vasodilated in response to BK, such vasodilation was less dependent on NO at HA than at LA. Furthermore, the responses to the NO donor SNP were attenuated in HA compared with LA MA, suggesting that the reduced vasodilator response to ACh in HA MA was due to impaired downstream NO signaling.

Our results were consistent with several lines of evidence indicating that hypoxia is not only a consequence of impaired vasodilation but also contributes to its etiology. We and others have reported the pregnancy complications of preeclampsia and IUGR to be increased in populations living at HA where ambient partial pressure of oxygen is 20–50% lower than at sea level depending on the elevation.1, 3, 21 Although the incidence of preeclampsia is increased at HA, its week of onset is not different from LA in Colorado.3 Furthermore, severe preeclampsia at HA accounts for a greater proportion (21%) of intrauterine deaths at HA than LA in Bolivia,22 but whether the incidence of severe preeclampsia is increased at HA is unknown. Moreover, IUGR and symptoms of preeclampsia can be replicated in animal models of pregnancy exposed to chronic hypoxia.16, 23, 24

Our findings were also consistent with previous studies on the effects of chronic hypoxia exposure in uterine arteries from pregnant experimental animals. For instance, while chronic hypoxia did not change uterine artery vasoconstrictor response to PE25 or vasodilator responses to ACh or BK in pregnant guinea pigs, there was less NO-dependent vasodilation at HA vs. LA.17 In rats exposed to hypoxia, NO-dependent cholinergic uterine-artery vasodilation was also diminished without affecting vasoconstrictor responses.16 Similarly, chronic hypoxia elevated myogenic tone in ovine uterine arteries by inhibiting large-conductance Ca2+-activated K+ (BKCa) channel activity via increased oxidative stress, upregulation of protein kinase C (PKC), and reduction of BKCa β1 subunit expression.10, 12, 26 Since in human umbilical vessels, ACh induces vasoconstriction by a mechanism involving increased PKC activity and reduction of smooth muscle BKCa channels,27 it is possible that the blunted vasodilator response to ACh in HA MA is due to reduced BKCa activation.

Human MA from pregnancies complicated by IUGR and preeclampsia compared with normal pregnancies have decreased MA lumenal diameters;28 blunted vasodilator responses to ACh,20 BK6, 18, 29 and shear stress;5 and increased U46619-induced oscillations in vascular tone.30 Our study aligns with these observations and indicates that hypoxia due to residence at HA, without the other pathophysiological characteristics of preeclampsia and/or IUGR, impairs the vasodilator response to ACh in otherwise healthy women. Moreover, since MA were not obtained from the site of placentation, the changes in MA vasodilator response observed were not a function of trophoblast-induced spiral arteriolar or MA remodeling.31 Because the hypoxia present in preeclamptic or IUGR pregnancies is likely local in nature, whereas global hypoxia is present under the circumstances of HA pregnancy, future studies are warranted to determine whether IUGR pregnancies at HA have further impaired vasodilation of MA and whether their mechanisms are similar to those from IUGR pregnancies at LA.

Given the lesser dependence on NO-induced vasodilation seen in the HA vs. the LA MA in our study, we asked whether there was less expression of eNOS protein. Because eNOS expression is restricted to endothelial cells in myometrial tissue,32 we were able to measure its expression and activation by phosphorylation of Ser-1177 in whole in myometrial tissue. By using CD31 rather than total myometrial protein, we were able to normalize the amount of eNOS protein expression and activation relative to the total amount of protein present in vascular tissue, thus overcoming the limitation imposed by the small amount of tissue available from MA alone. Further, since there might be more vessels in the particular section of myometrial tissue used, the use of CD31 effectively controlled for the number of vessels present. Although we found no differences in basal levels of total eNOS or p-eNOS in LA vs. HA MA, we cannot discard the possibility that differences could exist under conditions of ACh, BK, or other stimulation.

We did not find differences between the two altitudes in eNOS expression or phosphorylation. This contrasts to what has been reported in 4th generation uterine arteries from chronically-hypoxic ovine pregnancies where upregulation of eNOS expression and increased Ca2+-dependent NO production has been observed.33 However a reduction in birth weight was not seen in such studies,34 suggesting that upregulation of eNOS in the uterine vascular bed may have been a mechanism for protecting against hypoxia-associated IUGR. Alternatively, such differences in study results could be due to variation among species and/or vascular beds in the effects of hypoxia.

Rather our studies indicated that the mechanisms by which chronic hypoxia decreased MA vasodilator response and NO-dependence were downstream of NO production. Specifically, whereas the NO-donor SNP prompted MA vasodilation, the maximal vasodilation achieved was reduced in MA from HA compared to LA. However, because the expression and activation of eNOS in myometrial vessels was measured under unstimulated conditions and the reduced maximal vasodilation to SNP seen at HA was not as marked as the decrease in ACh response, we cannot exclude the possibility that other mechanisms may also be contributing to the lack of ACh vasodilation at HA. Such mechanisms may include: a) attenuation of muscarinic acetylcholine receptor(s) expression or function, b) reduced Ca2+-dependent eNOS activation as has been shown in pulmonary arteries from chronically-hypoxic sheep,35 and c) the possibility that differences in eNOS activity might be present under stimulated conditions. Therefore, future studies assessing expression and function of muscarinic acetylcholine receptors, Ca2+-dependent eNOS activation and NO production under stimulated conditions are necessary to elucidate the mechanisms by which HA impairs ACh vasodilation.

As summarized in Figure 5, we propose that while both ACh and BK vasodilate MA in an endothelial- and NO-dependent manner at LA, the chronic hypoxia of HA alters endothelial NO-dependent pathways to reduce ACh-elicited vasodilation of MA. The vasodilator response to BK is unaffected since BK can act through NO and/or endothelium-independent mechanisms by, for example, increasing production of vasodilator prostaglandins or endothelial-derived hyperpolarization factor (EDHF); however, the contribution of NO to BK vasodilation is still impaired in HA MA. Further, we propose that the mechanism by which NO signaling is disrupted in HA MA is not due to the lack of basal eNOS activation, as its phosphorylation at Ser-1177 was unchanged, but by the reduction in downstream effectors, such as sGC/PKG pathways, as evidenced by the attenuated relaxation in response to the exogenous NO donor SNP.

Figure 5. Chronic hypoxia of high-altitude residence switches vasodilatory pathways in MA from pregnant women.

Figure 5.

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At low altitude, MA are vasodilated by acetylcholine (ACh) and bradykinin (BK) acting largely through endothelial cell (EC)-dependent mechanisms. ACh and BK activate endothelial nitric oxide (NO) synthase (eNOS) by phosphorylation of Ser-1177 to produce NO, which diffuses to the smooth muscle cells (SMC) where it activates soluble guanylate cyclase (sGC) to increase cyclic GMP (cGMP) levels, and in turn, promote protein kinase G (PKG) activity, relaxation of the SMC and vasodilation of the MA. BK also acts through an unknown, largely endothelium-independent mechanism to induce relaxation of SMC. At high altitude, even though basal Ser-1177 phosphorylation of eNOS remains unchanged, decreased vasodilator response to the NO donor SNP suggests that the activation of the downstream NO pathway (i.e., sGC-cGMP-PKG) is reduced and thereby impairs ACh vasodilation of MA. Conversely, BK vasodilates MA at high altitude partially by activating cyclooxygenase (COX)-dependent production of prostaglandins (PGs), but also by two other putative mechanisms: an endothelium-dependent (likely endothelial-derived hyperpolarizing factor, EDHF) and an unknown, largely endothelium-independent mechanism. Other abbreviations: mAChR, muscarinic ACh receptor; B1/2, bradykinin receptor B1 or B2; L-arg, L-arginine, GTP, guanosine triphosphate; AA, arachidonic acid.

In summary, we concluded that our data indicated that chronic hypoxia reduced NO-dependent vasodilation in MA, which probably, in turn, contributes to raising uterine vascular resistance, lowering uterine artery blood flow and thereby hypoxia-related fetal growth restriction.

Perspectives

Our data demonstrate that HA diminished cholinergic-dependent vasodilation of MA without changing the MA vasoconstrictor response or myometrial vascularity and was due, in part, to a reduction in NO signaling and was accompanied by a decrease in birth weight. These studies contribute to our understanding of the effects of chronic hypoxia on uterine vascular responses to pregnancy in a setting where fetal growth restriction occurs. Because hypoxia characterizes IUGR or preeclampsia at any altitude, understanding the mechanisms by which hypoxia impairs myometrial vascular function during pregnancy is essential for identifying therapeutic targets for improving the treatments for women and their infants suffering from such hypoxia-related pregnancy complications.

Supplementary Material

Author's last name changed
Supplemental Material

Novelty and Significance:

1). What Is New

  • Chronic hypoxia alone (in the absence the other pathophysiology) impairs cholinergic vasodilation in myometrial arteries from healthy pregnant women.

  • This reduced cholinergic response is due to diminished NO signaling.

  • Vasodilation by bradykinin is not affected by altitude, but its mechanism of action is changed from being NO-dependent to NO-independent.

2). What Is Relevant?

  • High-altitude populations have an increased incidence of preeclampsia and IUGR.

  • Chronic hypoxia is related to the etiology of preeclampsia and IUGR at any altitude.

  • Impaired vasodilatory responses of myometrial arteries have been associated with the development of preeclampsia and IUGR.

Acknowledgments

We thank the University of Colorado Denver (UCD) Perinatal Clinical and Translational Research Center and the Obstetrics Research teams for their help in consenting and collecting tissues from the low-altitude subjects. Special thanks are extended to the St. Anthony Summit Medical Center nurses and doctors for their help in consenting high-altitude subjects and collecting myometrial tissue. In addition, Dr. K. Joseph Hurt provided lysates from wild type and eNOS knockout mouse lung, the UCD Research Histology Shared Resource assisted with the preparation of immunohistochemistry slides, and the UCD BA/BS-MD program supported research rotations of undergraduate students.

Source(s) of Funding

This work was supported by NIH R01-HD088590 (LGM and CGJ) and Center for Women’s Health Research Junior Faculty Seed (RAL) grants.

Footnotes

Conflict(s) of Interest/Disclosure(s)

None.

REFERENCES

  • 1.Julian CG, Galan HL, Wilson MJ, Desilva W, Cioffi-Ragan D, Schwartz J, Moore LG. Lower uterine artery blood flow and higher endothelin relative to nitric oxide metabolite levels are associated with reductions in birth weight at high altitude. Am J Physiol Regul Integr Comp Physiol. 2008;295:R906–915 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Moore LG, Niermeyer S, Zamudio S. Human adaptation to high altitude: Regional and life-cycle perspectives. Am J Phys Anthropol. 1998;Suppl 27:25–64 [DOI] [PubMed] [Google Scholar]
  • 3.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 1999;180:1161–1168 [DOI] [PubMed] [Google Scholar]
  • 4.Cockell AP, Poston L. Flow-mediated vasodilatation is enhanced in normal pregnancy but reduced in preeclampsia. Hypertension. 1997;30:247–251 [DOI] [PubMed] [Google Scholar]
  • 5.Kublickiene KR, Lindblom B, Kruger K, Nisell H. Preeclampsia: Evidence for impaired shear stress-mediated nitric oxide release in uterine circulation. Am J Obstet Gynecol. 2000;183:160–166 [DOI] [PubMed] [Google Scholar]
  • 6.Luksha L, Luksha N, Kublickas M, Nisell H, Kublickiene K. Diverse mechanisms of endothelium-derived hyperpolarizing factor-mediated dilatation in small myometrial arteries in normal human pregnancy and preeclampsia. Biol Reprod. 2010;83:728–735 [DOI] [PubMed] [Google Scholar]
  • 7.Konje JC, Howarth ES, Kaufmann P, Taylor DJ. Longitudinal quantification of uterine artery blood volume flow changes during gestation in pregnancies complicated by intrauterine growth restriction. BJOG. 2003;110:301–305 [PubMed] [Google Scholar]
  • 8.Browne VA, Toledo-Jaldin L, Davila RD, Lopez LP, Yamashiro H, Cioffi-Ragan D, Julian CG, Wilson MJ, Bigham AW, Shriver MD, Honigman B, Vargas E, Roach R, Moore LG. High-end arteriolar resistance limits uterine artery blood flow and restricts fetal growth in preeclampsia and gestational hypertension at high altitude. Am J Physiol Regul Integr Comp Physiol. 2011;300:R1221–1229 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Rockwell LC, Keyes LE, Moore LG. Chronic hypoxia diminishes pregnancy-associated DNA synthesis in guinea pig uteroplacental arteries. Placenta. 2000;21:313–319 [DOI] [PubMed] [Google Scholar]
  • 10.Hu XQ, Xiao D, Zhu R, Huang X, Yang S, Wilson SM, Zhang L. Chronic hypoxia suppresses pregnancy-induced upregulation of large-conductance ca2+-activated k+ channel activity in uterine arteries. Hypertension. 2012;60:214–222 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Xiao D, Longo LD, Zhang L. Role of katp and l-type ca2+ channel activities in regulation of ovine uterine vascular contractility: Effect of pregnancy and chronic hypoxia. Am J Obstet Gynecol 2010;203:596 e596–512 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Xiao D, Zhu R, Zhang L. Gestational hypoxia up-regulates protein kinase c and inhibits calcium-activated potassium channels in ovine uterine arteries. Int J Med Sci. 2014;11:886–892 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhu R, Hu XQ, Xiao D, Yang S, Wilson SM, Longo LD, Zhang L. Chronic hypoxia inhibits pregnancy-induced upregulation of skca channel expression and function in uterine arteries. Hypertension. 2013;62:367–374 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Xiao D, Hu XQ, Huang X, Zhou J, Wilson SM, Yang S, Zhang L. Chronic hypoxia during gestation enhances uterine arterial myogenic tone via heightened oxidative stress. PLoS One. 2013;8:e73731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.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. 2003;284:H820–829 [DOI] [PubMed] [Google Scholar]
  • 16.Aljunaidy MM, Morton JS, Cooke CL, Davidge ST. Maternal vascular responses to hypoxia in a rat model of intrauterine growth restriction. Am J Physiol Regul Integr Comp Physiol. 2016;311:R1068–R1075 [DOI] [PubMed] [Google Scholar]
  • 17.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. 2000;278:H2069–2075 [DOI] [PubMed] [Google Scholar]
  • 18.Ong SS, Moore RJ, Warren AY, Crocker IP, Fulford J, Tyler DJ, Gowland PA, Baker PN. Myometrial and placental artery reactivity alone cannot explain reduced placental perfusion in pre-eclampsia and intrauterine growth restriction. BJOG. 2003;110:909–915 [PubMed] [Google Scholar]
  • 19.Clark AR, James JL, Stevenson GN, Collins SL. Understanding abnormal uterine artery doppler waveforms: A novel computational model to explore potential causes within the utero-placental vasculature. Placenta. 2018;66:74–81 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Wimalasundera RC, Thom SA, Regan L, Hughes AD. Effects of vasoactive agents on intracellular calcium and force in myometrial and subcutaneous resistance arteries isolated from preeclamptic, pregnant, and nonpregnant woman. Am J Obstet Gynecol. 2005;192:625–632 [DOI] [PubMed] [Google Scholar]
  • 21.Giussani DA, Phillips PS, Anstee S, Barker DJ. Effects of altitude versus economic status on birth weight and body shape at birth. Pediatr Res. 2001;49:490–494 [DOI] [PubMed] [Google Scholar]
  • 22.Keyes LE, Armaza JF, Niermeyer S, Vargas E, Young DA, Moore LG. Intrauterine growth restriction, preeclampsia, and intrauterine mortality at high altitude in bolivia. Pediatr Res. 2003;54:20–25 [DOI] [PubMed] [Google Scholar]
  • 23.Skeffington KL, Higgins JS, Mahmoud AD, Evans AM, Sferruzzi-Perri AN, Fowden AL, Yung HW, Burton GJ, Giussani DA, Moore LG. Hypoxia, ampk activation and uterine artery vasoreactivity. J Physiol. 2016;594:1357–1369 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Thompson LP, Pence L, Pinkas G, Song H, Telugu BP. Placental hypoxia during early pregnancy causes maternal hypertension and placental insufficiency in the hypoxic guinea pig model. Biol Reprod. 2016;95:128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.White MM, McCullough RE, Dyckes R, Robertson AD, Moore LG. Effects of pregnancy and chronic hypoxia on contractile responsiveness to alpha1-adrenergic stimulation. J Appl Physiol (1985). 1998;85:2322–2329 [DOI] [PubMed] [Google Scholar]
  • 26.Hu XQ, Huang X, Xiao D, Zhang L. Direct effect of chronic hypoxia in suppressing large conductance ca(2+)-activated k(+) channel activity in ovine uterine arteries via increasing oxidative stress. J Physiol. 2016;594:343–356 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Chen N, Lv J, Bo L, Li N, Wu C, Yin X, Li J, Tao J, Chen J, He Y, Huang S, Xiao J, Mao C, Xu Z. Muscarinic-mediated vasoconstriction in human, rat and sheep umbilical cords and related vasoconstriction mechanisms. BJOG. 2015;122:1630–1639 [DOI] [PubMed] [Google Scholar]
  • 28.Ong SS, Baker PN, Mayhew TM, Dunn WR. Remodeling of myometrial radial arteries in preeclampsia. Am J Obstet Gynecol. 2005;192:572–579 [DOI] [PubMed] [Google Scholar]
  • 29.Moyes AJ, Gray GA, Denison FC. Bradykinin b1 receptor-mediated vasodilation is impaired in myometrial arteries from women with pre-eclampsia. Hypertens Pregnancy. 2014;33:177–190 [DOI] [PubMed] [Google Scholar]
  • 30.Sweeney M, Wareing M, Mills TA, Baker PN, Taggart MJ. Characterisation of tone oscillations in placental and myometrial arteries from normal pregnancies and those complicated by pre-eclampsia and growth restriction. Placenta. 2008;29:356–365 [DOI] [PubMed] [Google Scholar]
  • 31.Lyall F Priming and remodelling of human placental bed spiral arteries during pregnancy--a review. Placenta. 2005;26 Suppl A:S31–36 [DOI] [PubMed] [Google Scholar]
  • 32.Bartlett SR, Bennett PR, Campa JS, Dennes WJ, Slater DM, Mann GE, Poston L, Poston R. Expression of nitric oxide synthase isoforms in pregnant human myometrium. J Physiol. 1999;521 Pt 3:705–716 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.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. 2001;280:H812–820 [DOI] [PubMed] [Google Scholar]
  • 34.Kamitomo M, Longo LD, Gilbert RD. Right and left ventricular function in fetal sheep exposed to long-term high-altitude hypoxemia. Am J Physiol. 1992;262:H399–405 [DOI] [PubMed] [Google Scholar]
  • 35.Blum-Johnston C, Thorpe RB, Wee C, Opsahl R, Romero M, Murray S, Brunelle A, Blood Q, Wilson R, Blood AB, Zhang L, Longo LD, Pearce WJ, Wilson SM. Long-term hypoxia uncouples ca(2+) and enos in bradykinin-mediated pulmonary arterial relaxation. Am J Physiol Regul Integr Comp Physiol. 2018;314:R870–R882 [DOI] [PMC free article] [PubMed] [Google Scholar]

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