Skip to main content
NCBI home page
The Journal of Physiology logoLink to The Journal of Physiology
. 2015 Jul 31;594(5):1357–1369. doi: 10.1113/JP270995

Hypoxia, AMPK activation and uterine artery vasoreactivity

K L Skeffington 1, J S Higgins 1, A D Mahmoud 2, A M Evans 2, A N Sferruzzi‐Perri 1, A L Fowden 1, H W Yung 1, G J Burton 1, D A Giussani 1, L G Moore 3,✉
PMCID: PMC4771787  PMID: 26110512

Key points

  • Uterine artery vasodilatation is a key mechanism for increasing utero‐placental blood flow and fetal nutrient supply.

  • Since the pioneering work of Joseph Barcroft, the natural laboratory of high altitude has been used to study the mechanisms regulating uterine artery blood supply and fetal growth.

  • Genes near the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) have been implicated in genetic protection from high altitude‐associated fetal growth restriction.

  • We show that AMPK is present in utero‐placental tissues and has vasodilator effects in murine uterine arteries, and that exposure to chronic hypoxia sufficient to decrease fetal growth increases the vasodilator actions of AMPK in opposing phenylephrine‐induced vasoconstriction.

  • These results point to AMPK as being a key link between maternal vascular responses to pregnancy and fetal growth. Manipulation of AMPK may be a novel mechanism for developing new therapies in pregnancies complicated by chronic hypoxia.

Abstract

Genes near adenosine monophosphate‐activated protein kinase‐α1 (PRKAA1) have been implicated in the greater uterine artery (UtA) blood flow and relative protection from fetal growth restriction seen in altitude‐adapted Andean populations. Adenosine monophosphate‐activated protein kinase (AMPK) activation vasodilates multiple vessels but whether AMPK is present in UtA or placental tissue and influences UtA vasoreactivity during normal or hypoxic pregnancy remains unknown. We studied isolated UtA and placenta from near‐term C57BL/6J mice housed in normoxia (n = 8) or hypoxia (10% oxygen, n = 7–9) from day 14 to day 19, and placentas from non‐labouring sea level (n = 3) or 3100 m (n = 3) women. Hypoxia increased AMPK immunostaining in near‐term murine UtA and placental tissue. RT‐PCR products for AMPK‐α1 and ‐α2 isoforms and liver kinase B1 (LKB1; the upstream kinase activating AMPK) were present in murine and human placenta, and hypoxia increased LKB1 and AMPK‐α1 and ‐α2 expression in the high‐ compared with low‐altitude human placentas. Pharmacological AMPK activation by A769662 caused phenylephrine pre‐constricted UtA from normoxic or hypoxic pregnant mice to dilate and this dilatation was partially reversed by the NOS inhibitor l‐NAME. Hypoxic pregnancy sufficient to restrict fetal growth markedly augmented the UtA vasodilator effect of AMPK activation in opposition to PE constriction as the result of both NO‐dependent and NO‐independent mechanisms. We conclude that AMPK is activated during hypoxic pregnancy and that AMPK activation vasodilates the UtA, especially in hypoxic pregnancy. AMPK activation may be playing an adaptive role by limiting cellular energy depletion and helping to maintain utero‐placental blood flow in hypoxic pregnancy.

Key points

  • Uterine artery vasodilatation is a key mechanism for increasing utero‐placental blood flow and fetal nutrient supply.

  • Since the pioneering work of Joseph Barcroft, the natural laboratory of high altitude has been used to study the mechanisms regulating uterine artery blood supply and fetal growth.

  • Genes near the metabolic sensor adenosine monophosphate‐activated protein kinase (AMPK) have been implicated in genetic protection from high altitude‐associated fetal growth restriction.

  • We show that AMPK is present in utero‐placental tissues and has vasodilator effects in murine uterine arteries, and that exposure to chronic hypoxia sufficient to decrease fetal growth increases the vasodilator actions of AMPK in opposing phenylephrine‐induced vasoconstriction.

  • These results point to AMPK as being a key link between maternal vascular responses to pregnancy and fetal growth. Manipulation of AMPK may be a novel mechanism for developing new therapies in pregnancies complicated by chronic hypoxia.

Abbreviations

AICAR

5‐amino‐1‐β‐d‐ribofuranosyl‐imadazole‐4‐carboxamide

AMPK

AMP‐activated protein kinase

AUC

area under the curve

BKCa

large‐conductance potassium channel

BPD

biparietal diameter

DAB

3,3′‐diaminobenzidine

JZ

junctional zone

KCl

potassium chloride

LKB1

liver kinase B1

LZ

labyrinthine zone

mTOR

mechanistic target of rapamycin

NO

nitric oxide

NOS

nitric oxide synthase

PE

phenylephrine

PRKAA1

the gene coding for AMPK‐α1 isoform

THR

threonine

UtA

uterine artery

Introduction

Pioneering studies of Joseph Barcroft and his students drew attention to the value of high altitude as a natural laboratory for understanding the mechanisms by which intrauterine hypoxia reduces fetal growth (Barcroft, 1933; Barron et al. 1964). A key determinant of fetal growth is the pregnancy rise in uterine artery (UtA) blood flow, which is due, in turn, to profound changes in multiple physiological systems (Gant & Worley, 1989). Among the greatest are the structural remodelling and alterations in vasoreactivity of the uterine vasculature that result in approximately 20% of the maternal cardiac output being directed to the utero‐placental circulation by term (Osol & Moore, 2014). Isolated vessel studies in experimental animals show that chronic hypoxia is associated with a decreased vasodilator response to flow and to pharmacological agonists in the main UtA and downstream vessels (White et al. 2000; Mateev et al. 2003; Xiao et al. 2010), which may, in turn, contribute to the reduced UtA blood flow and altitude‐associated fetal growth restriction seen in high‐altitude newcomers (Zamudio et al. 1995; Julian et al. 2008). Multigenerational highland residents (Andeans and Tibetans) are relatively protected from altitude‐associated reductions in fetal growth and have a greater pregnancy rise in UtA blood flow compared with altitude newcomers (Moore et al. 1998, 2001; Giussani et al. 2001; Chen et al. 2002; Julian et al. 2007, 2009; Soria et al. 2013). Such protection appears to be due, in part, to genetic factors given that it is proportional to the degree of highland ancestry and is not the result of the woman's own duration of residence at high altitude (Bennett et al. 2008; Julian et al. 2011; Soria et al. 2013).

Identifying the genes and signalling pathways involved in protecting native highlanders presents a novel means for addressing the mechanisms by which intrauterine hypoxia influences fetal growth. Several gene regions have been acted upon by natural selection in long‐resident populations (Bigham et al. 2009, 2010; Beall et al. 2010; Simonson et al. 2010; Yi et al. 2010; Alkorta‐Aranburu et al. 2012). In Andeans prominent among these are single nucleotide polymorphisms (SNPs) near adenosine monophosphate‐activated protein kinase α1 (PRKAA1) (Bigham et al. 2009, 2010). Further, the PRKAA1 variants more common in Andeans are positively associated with infant birth weight as well as with key determinants of fetal growth, namely the pregnancy‐associated increase in UtA diameter and the expression patterns of genes in metabolic pathways proposed to play a role in altitude‐associated fetal growth restriction (Yung et al. 2012; Bigham et al. 2014).

AMPK is a ubiquitously expressed enzyme in eukaryotes that is stimulated by stresses that deplete cellular ATP and thus serves as a metabolic sensor for matching tissue energy demand with supply (Hardie et al. 2012). It is composed of three subunits (α, β, γ), each of which has multiple isoforms whose expression levels vary by tissue type (Viollet et al. 2010). The α1 and α2 isoforms are the catalytic subunits and contain the Thr‐172 site where AMPK is activated more than 100‐fold via phosphorylation by liver kinase B1 (LKB1) and other upstream kinases (Evans et al. 2009; Viollet et al. 2009). In skeletal muscle AMPK activation promotes glucose uptake and mitochondrial biogenesis, and decreases energy demand by inhibiting the mechanistic target of rapamycin (mTOR) and switching on various catabolic enzymes (Hardie, 2011). More recently AMPK activation has been recognized to have vascular effects, acting to stimulate endothelial nitric oxide (NO) production (Wang & Proud, 2006) as well as to regulate smooth muscle function directly (Goirand et al. 2007). Underscoring its importance under conditions of hypoxia, AMPK activation has been implicated in the aetiology of early‐onset pre‐eclampsia (Yung et al. 2014 a) cardiorespiratory responses to hypoxia (Evans, 2006), and hypoxic pulmonary vasoconstriction (Evans et al. 2005).

It is unknown whether AMPK activation influences UtA vasoreactivity during pregnancy and, if so, whether such effects are altered by exposure to hypoxia. We used a broad‐ranging approach to address such questions in which human as well as experimental‐animal tissues were studied using multiple methods. Specifically, we hypothesized that AMPK is present in UtA and placental tissue and that its expression was increased during pregnancy by exposure to hypoxia as detected using immunohistochemistry in mice and quantitative RT‐PCR in previously collected human tissues. We further hypothesized that AMPK activation prompted UtA vasodilatation and/or altered vasoconstrictor sensitivity to phenylephrine (PE) in vessels isolated from near‐term mice, and that such effects were altered by exposure to hypoxia. To test these hypotheses we used the pharmacological AMPK agonist A769662 and compared UtA vasodilator and vasoconstrictor responses in vessels isolated from the normoxic vs. hypoxic animals. Finally, we treated vessels with the NOS inhibitor N G‐nitro‐l‐arginine methyl ester (l‐NAME) to determine the contribution of NO‐dependent and ‐independent mechanisms to the effects of AMPK activation observed. We considered that such studies would improve our understanding of the mechanisms regulating maternal vascular responses to pregnancy and fetal growth under conditions of chronic hypoxia.

Methods

Ethical approval

All mouse experiments were carried out using procedures consistent with the UK Animals (Scientific Procedures) Act 1986 and approved by the Local Ethics Review Committee of the University of Cambridge. Placentas were collected from human subjects who provided written informed consent to procedures approved by the University of Colorado Multiple Institutional Review Board (COMIRB, Aurora, CO, USA), the institutional review board of University College Hospital London (London, UK), and the Cambridge Local Research Ethics Committee and which conformed to the Declaration of Helsinki.

Samples and protocols

Female C57BL/6J mice were placed with males overnight. The presence of a copulatory plug the following morning was taken to indicate day 1 of pregnancy. Pregnant mice (n = 31) were housed in groups of two or three in rooms with 21% O2, 12 h light–dark cycles and controlled temperature (21°C) and humidity (60%). They had ad libitum access to food (Rat and Mouse No. 3 Breeding, Special Diet Services, Witham, UK). At day 14 of pregnancy (term is ∼21 days), animals were randomly assigned to normoxic or hypoxic (10% O2) treatment groups. Hypoxia was achieved by placing the animals in a chamber containing a PVC isolator (PFI Systems, Milton Keynes, UK) and a N2 generator (N2MID60, Domnick Hunter Ltd, UK) so as to control the percentage O2 within the chamber, without changing the CO2 levels, by altering the inflows of air and N2. O2 levels were monitored using an O2 analyser (ICA, UK) and CO2 levels by a portable CO2 analyser that was calibrated daily (The Electronic Workshop, Department of Physiology, Development and Neuroscience, University of Cambridge). Normoxic animals were housed in the same room that contained the chambers. Maternal weight, food and water intake were monitored daily. This was achieved in the hypoxic group via a sealed transfer box that could be opened briefly without altering O2 levels within the main chamber.

On day 19 of pregnancy mice were killed by cervical dislocation. The uterus was dissected immediately, the numbers of viable fetuses and fetal reabsorptions counted, fetal and placental weights recorded, and fetal biometry taken. Both UtAs were removed, placed in ice cold phosphate‐buffered saline (PBS), and processed immediately (<5 min) for either myography or immunohistochemistry. In approximately half the normoxic and hypoxic animals, one placenta whose weight was closest to the litter mean was prepared for immunohistochemistry.

Samples from human placentas were obtained from term, non‐labouring women residing either at sea level (n = 3) or high altitude (3100 m, n = 3) as described previously (Yung et al. 2012).

Immunohistochemistry

The murine uterine vessels were placed in ice cold PBS, cleared of adipose tissues and the uterine horn using a dissecting microscope (Leica, Germany), and fixed in 4% paraformaldehyde. The main UtA together with its first and second order branches was divided into two to three longitudinal segments, embedded in a single paraffin block, and sectioned. Whole placentas were washed with PBS, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Paraffin‐embedded sections were de‐waxed, washed, prepared for antigen retrieval using 0.01 m citric buffer (pH 6.0), and incubated overnight using either primary antibody (phospho‐AMPKα [Thr172] [40H9] rabbit) from Cell Signaling Technology (Hitchin, UK) diluted in 5% goat serum (GS)/horse serum (HS) in Tris‐buffered saline (TBS) or vehicle alone. The following day, the secondary antibody (anti‐rabbit IgG, diluted 1:200 in 5% GS/HS) was applied, incubated for 1 h, developed for staining using DAB and counterstained with haematoxylin. The slides were scanned using a Nanozoomer (Hamamatsu Photonics, Welwyn Garden City, UK), saved as high‐resolution files, and images quantified using IPLAb software (v6.0, Scanalytics, Fairfax, VA, USA). 3,3′‐Diaminobenzidine (DAB)‐positive areas were selected and segmented in a region of interest based on a selective distribution of saturation and hue values that matched the colour of the DAB reactive product and the pattern of staining of the image. Segmented overlays representing the selected pixels of DAB staining were selected in individual UtAs and the labyrinthine and junctional zones (separately), quantified and expressed as the percentage area of positive DAB staining per area of tissue examined.

Quantitative RT‐PCR

RNA from mouse (n = 3) or human (n = 6) placental tissue was extracted using the miRNeasy Mini Kit from Qiagen (Manchester, UK) following the manufacturer's guidelines, and the concentration determined using a Nanodrop 1000 spectrophotometer (Thermo Scientific, Hemel Hempstead, UK). cDNA synthesis was carried out using the Transcriptor High Fidelity cDNA synthesis Kit (Roche, UK) following the manufacturer's instructions. For qPCR analysis, 2.5 μl of cDNA in RNase free water was made up to 25 μl with FastStart Universal SYBR Green Master (ROX, 12.5 μl, Roche), Ultra Pure Water (8 μl, Sigma, UK) and forward and reverse primers for LKB1, AMPK‐α1 and AMPK‐α2 (Qiagen, UK). The sample was then centrifuged and 25 μl added to a MicroAmpTM Fast Optical 96‐Well Reaction Plate (Greiner Bio‐One, Stonehouse, UK), the reaction plate was sealed with an optical adhesive cover (Applied Biosystems, Warrington, UK) and the plate was centrifuged. The reaction was then run on a sequence detection system (Applied Biosystems) using AmpliTaq Fast DNA Polymerase with a 2 min initial step at 50°C followed by a 10 min step at 95°C, a 15 s step at 95°C (repeated 40 times) followed by a dissociation stage with a 15 s step at 95°C, and followed by a 20 s at 60°C and a 15 s step at 95°C. Negative controls included tissue aspirants for which no reverse transcriptase was added, and aspiration of extracellular medium and PCR controls. None of the controls produced any detectable amplicon, ruling out genomic or other contamination.

Myography

The murine uterine vasculature was dissected and pinned out in a dish of ice‐cold Krebs solution (in mmol l−1, all reagents from Sigma UK unless specified otherwise: NaCl 118.5, NaHCO3 25, KCl 4.75, MgSO4.7H2O 1.2, KH2PO4 1.2, CaCl2 2.5, d‐glucose 11.1). Using a bifocal dissecting microscope (Brunel Microscopes Ltd, Chippenham, UK), first‐order 2 mm in length UtA segments were dissected and cleared of any connective or adipose tissue. The vessel segments were then mounted in a four‐chamber, small‐vessel wire myograph (Multi Wire Myograph System 620M, DMT, Aarhus, Denmark) by threading two wires 40 μm in diameter through the vessel lumen and attaching one to a pressure transducer and the other to a micrometer as previously described (Pulgar et al. 2011; Giussani et al. 2012). The chamber was continually gassed with 5% CO2 and 95% O2 to maintain a physiological pH, and gradually heated to 37°C. Throughout the investigation, the Krebs solution was refreshed every 20 min, and the vessels allowed to re‐equilibrate for at least 20 min between experiments.

UtAs were normalized to 0.9 of L 13.3kPa , or the internal circumference at which active force production and sensitivity to agonists is maximal, expressed in kilo pascals and allowed to equilibrate for 20 min. Following normalization a ‘wake up’ protocol was performed consisting of exposure of the vessels to a high concentration of potassium (KCl, 125 mmol l−1). Vessel viability was verified by a positive constrictor response to phenylephrine (PE, 1 × 10−4 m) and a positive relaxant response to acetylcholine (ACh, 1 × 10−10 m to 1 × 10−4 m). Two vessels from each animal were pre‐constricted with a dose of PE determined to produce 70% of the maximal constriction to PE for each vessel. The submaximally pre‐constricted vessels were then relaxed by addition of the AMPK agonist A769662 (Tocris, USA) dissolved in DMSO in increasing concentrations (1 × 10−6 m to 1 × 10−4 m) at 10 min intervals, and the results from the two vessels per animal averaged. We chose A769662 because, unlike 5‐amino‐1‐β‐d‐ribofuranosyl‐imidazole‐4‐carboxamide (AICAR) or metformin, it activates AMPK directly, and not other enzymes that are responsive to AMP levels (Goransson et al. 2007). Another advantage is that A769662, unlike AICAR, is not taken up by the adenosine transporter and therefore does not lead to the accumulation of adenosine outside cells or necessitate the use of an adenosine antagonist (Goransson et al. 2007). A third and fourth vessel segment from each animal was also tested using increasing doses of PE (1 × 10−9–1 × 10−4 m), with this then being repeated following 20 min incubation with A769662 (1 × 10−4 m) and finally repeated a third time following incubation with A769662 (1 × 10−4 m) plus the NOS inhibitor l‐NAME (1 × 10−5 m). The finding that the third curve was consistently larger than the second provided reassurance that the response of the vessels was not deteriorating over time. Studies were completed in vessels from a total of 17 animals.

Data and statistical analyses

The contractile response to PE was expressed as a percentage of the maximal constriction produced by KCl (% K max). The curves were fitted with the best‐fit line for describing the response to a given agonist. The negative logarithm to base 10 of the dose at which vessels were 50% maximally constricted to PE (EC50 or pD 2) was used as an index of vascular sensitivity, and the area under the curve (AUC) was calculated to assess overall reactivity using Prism v6.0 (GraphPad Software, La Jolla, CA, USA). Single values were compared between normoxic and hypoxic groups using Student's unpaired t test. Differences in immunostaining or mRNA expression between normoxic and hypoxic groups were assessed using one‐way ANOVA with the Student–Neuman–Keuls post hoc test. The effects of AMPK activation on PE‐induced constriction were assessed using two‐way ANOVA with Tukey's post hoc test (SigmaStat, Systat Software, London, UK). Data are presented as the means ± standard error of the mean (SEM). Significance was accepted when the two‐tailed P < 0.05 and reported as trends when 0.05 < P < 0.10.

Results

Maternal and fetal characteristics

Murine maternal body weights were similar at days 1 and 14, but reduced at day 19 in the hypoxic compared with normoxic groups (Table 1). Litter size was not reduced in the hypoxic group although there was a trend for a greater number of reabsorptions. Fetuses from hypoxic pregnancies weighed less, but placental weights were similar to those seen in the normoxic group. Fetuses from the hypoxic compared with the normoxic group had smaller crown‐rump lengths, biparietal diameters (BPDs) and a greater BPD to body weight ratio, indicating asymmetric growth restriction (Table 1).

Table 1.

Characteristics of the murine and human subjects

Variable Normoxic group Hypoxic group P
Mice
Sample size 8 7–9
Maternal body weight (g)
Day 1 26.4 ± 1.8 26.2 ± 0.8 0.89
Day 14 31.9 ± 2.2 31.8 ± 0.8 0.93
Day 19 37.4 ± 3.0 32.5 ± 1.1 0.046
Litter size (n) 6.0 ± 1.1 6.6 ± 0.4 0.59
Reabsorptions (n) 0.4 ± 0.2 1.2 ± 0.4 0.06
Fetal weight (mg) 1101.6 ± 11.0 816.2 ± 36.3 <0.0001
Placental weight (mg) 101.4 ± 7.5 95.5 ± 3.8 0.42
Fetal crown‐rump length (mm) 18.2 ± 0.3 16.3 ± 0.4 0.002
Fetal biparietal diameter (mm) 5.4 ± 0.2 4.8 ± 0.1 0.03
Fetal biparietal diameter/weight 0.49 ± 0.02 0.58 ± 0.02 0.006
UtA internal circumference (μm) 847.6 ± 34.1 911.8 ± 39 0.21
KCl maximum contraction (mN/mm) 1.08 ± 0.2 0.79 ± 0.1 0.20
PE maximal contraction (%Kmax) 124.8 ± 11.8 166.2 ± 17.5 0.07
pD 2 (m) 6.8 ± 0.29 7.1 ± 0.3 0.50
PE + A769662 pD 2 without l‐NAME (m) 5.7 ± 0.17 5.2 ± 0.1 0.045
PE + A769662 pD 2 with l‐NAME (m) 5.9 ± 0.16 5.6 ± 0.2 0.32
Human subjects
Sample size 3 3
Placental weight (g) 670 ± 118 530 ± 35 0.33
Infant birth weight (g) 3813 ± 104 3263 ± 502 0.58
Infant gestational age (weeks) 39.2 ± 1.4 39.8 ± 1.7 0.78
Open in a new tab

Values are means ± SEM. Bolded P values are <0.05. Abbreviations: KCl, potassium chloride; l‐NAME, N G‐nitro‐l‐arginine methyl ester hydrochloride; PE, phenylephrine; pD 2, negative logarithm to base 10 of the EC50 or the dose at which 50% of the maximal contraction was achieved.

Placental and birth weights were similar for the non‐labouring, sea‐level or high‐altitude women (Table 1), although birth weight was reduced at high altitude in the larger group of subjects from which these data were derived (Yung et al. 2012).

Immunohistochemistry

The uterine vessels from normoxic mice demonstrated staining for phosphorylated (activated) AMPK in arteries of varying size. The hypoxic compared with normoxic animals had greater staining intensity with primary antibody (22.0% vs. 11.0%, respectively, P < 0.0001) whereas the staining intensity did not differ in the vessels from normoxic animals with and without antibody (P = 0.12), indicating minimal AMPK expression (Fig. 1, left hand panels). Phosphorylated AMPK was also present in all regions of the mouse placenta (Fig. 1, right hand panels). Whereas staining intensity did not differ between the junctional zone (JZ) and labyrinthine zone (LZ) of the placenta from normoxic animals (P not significant), hypoxic exposure markedly increased LZ relative to JZ staining (P < 0.0001).

Figure 1. Phosphorylated AMPK (brown staining) is present in uterine arteries (UtA) and placentas from normoxic mice and increased by hypoxia in the UtA and the placental labyrinthine (LZ) but not junctional (JZ) or the decidua (DEC) zones .

Figure 1

Open in a new tab

For the placenta specimens, panels A and B are with and without AMPK antibody, respectively, at 5× and panels C and D are with AMPK antibody at 20× magnification.

Quantitative RT‐PCR

Results from quantitative RT‐PCR assays showed that mRNA for LKB1, AMPK‐α1 and AMPK‐α2 (as a percentage of β‐actin) were present in human and mouse placental tissue. Levels of RT‐PCR products for LKB1 and both AMPK catalytic isoforms were greater in the tissues from the high‐altitude compared with sea‐level human placentas (Fig. 2, top panel). In sea‐level mouse placentas, AMPK‐α2 levels were higher in the labyrinthine than junctional zones (Fig. 2, bottom panel).

Figure 2. mRNA expression relative to beta‐actin, in human and murine placentas .

Figure 2

Open in a new tab

Upper panel, mRNA for both AMPK α‐subunits and LKB1 is present at higher levels in human placentas from 3100 m vs. sea level. Lower panel, murine placentas also contain LKB1 and both AMPK α‐subunits at sea level, with expression levels for the α2‐subunit being greater in the labyrinthine than junctional zone. *P < 0.05, **P < 0.01, ****P < 0.0001. Data are means ± SEM.

Myography

The internal circumference of the isolated UtA at the time of study was similar in the normoxic and hypoxic murine groups (Table 1). There was no difference in the maximal contraction to KCl or PE, the contractile sensitivity to PE as measured by the pD 2 (Table 1), or the relaxation response to ACh (10−10 –10−4 m, P not significant, data not shown).

A769662, an AMPK agonist, caused complete, concentration‐dependent relaxation in the PE pre‐constricted UtA and that relaxation was similar in the normoxic and hypoxic mice (Fig. 3 A). Inhibition of NOS by incubation with l‐NAME reduced the relaxation response to A769662 by 30% but did not fully reverse it, indicating that 70% was NO independent in both the normoxic and hypoxic groups (Fig. 3 B).

Figure 3. Murine uterine artery relaxation in response to the pharmacological AMPK agonist A769662 .

Figure 3

Open in a new tab

A, uterine arteries pre‐constricted with a submaximal dose of PE from normoxic (n = 5) and hypoxic (n = 7) pregnant (day 19) mice relax in response to increasing concentrations of the AMPK agonist, A769662. B, the area under the curve (AUC), representing nitric oxide (NO)‐dependent and ‐independent components did not differ in the normoxic and hypoxic groups. Data are means ± SEM.

Incubation with the AMPK agonist A769662 reduced UtA contractile sensitivity to PE in both the normoxic and hypoxic mice as demonstrated by the rightward shifts in the contractile concentration–response curves (Fig. 4 A and B), with the change due to A769662 being greater in the hypoxic than normoxic groups (Fig. 4 C). Treatment of the vessel with l‐NAME increased contractile sensitivity to PE in vessels from normoxic or hypoxic mice (two‐way ANOVA, both P < 0.05), indicating that a portion of A769662's effect in opposing PE‐induced vasoconstriction was likely to be due to increased NO production or activity. However, l‐NAME treatment did not fully reverse A769662's effect, indicating that there was a NO‐independent contribution as well. Moreover the NO‐independent component was principally responsible for the greater vasodilator effect of AMPK activation seen in the vessels from hypoxic than normoxic animals (Fig. 4 D).

Figure 4. Murine uterine artery constriction to phenylephrine (PE) in control or A769662 treated vessels, with and without l‐NAME .

Figure 4

Open in a new tab

Incubation with A769662 (1 × 10−4 m, open circles, dotted lines) decreased contractile sensitivity to phenylephrine (PE) in UtA from both normoxic (A, n = 8) and hypoxic (B, n = 6–7) pregnant mice compared to values obtained prior to incubation with A769662 (open circles, continuous lines). This decrease was significant in both groups when expressed as the area under the curve (AUC) (C, P < 0.05, two way ANOVA and post‐hoc Tukey's test). The AUC, representing the change in sensitivity to PE following incubation with A769662, was significantly greater in the hypoxic compared to the normoxic animals (C, P < 0.05, unpaired t test). Treatment with l‐NAME (1 × 10−5 m, filled squares and dashed lines) decreased the contractile response to PE in A769662‐treated vessels from normoxic (A) and hypoxic (B) pregnant mice, indicating a significant NO‐dependent component (both P < 0.05), but did not fully restore the contractile response. The contributions of both nitric oxide (NO)‐dependent and ‐independent mechanisms as well as the total relaxation response were greater in the UtA from hypoxic compared with normoxic animals (D, P < 0.05, unpaired t test). *P < 0.05. Data are means ± SEM.

Discussion

Our principal findings were that AMPK was present in uterine arteries from near‐term mice as well as in mouse and human placental tissue, and that AMPK expression was increased by hypoxia as judged by immunohistochemistry and mRNA expression. Consistent with the possibility that the LKB1–AMPK signalling pathway influenced vascular reactivity, we found that pharmacological activation of AMPK caused PE‐preconstricted UtA from pregnant mice to relax due to both NO‐dependent and NO‐independent mechanisms. While the vasodilator effect of AMPK activation was similar in UtA isolated from normoxic or hypoxic mice, AMPK activation more markedly opposed PE‐induced constriction in the UtA from the hypoxic than the normoxic mice and this was due to NO‐dependent as well as to, largely, NO‐independent mechanisms. We therefore concluded that AMPK activation dilates uterine arteries, particularly in response to PE‐induced vasoconstriction, and that this latter effect is enhanced by hypoxic pregnancy.

AMPK activation in non‐pregnant animals has chiefly been studied for its metabolic effects. Drugs such as metformin, which is widely prescribed for lowering glucose levels in individuals with type 2 diabetes, decrease hepatic glucose production by mildly inhibiting the mitochondrial respiratory chain complex I, which in turn activates AMPK (Viollet et al. 2012). In skeletal muscle, AMPK activation promotes glucose uptake and mitochondrial biogenesis, and decreases energy demand by inhibiting the mechanistic target of rapamycin (mTOR) or by switching on the expression of various catabolic enzymes (Hardie, 2011). More recently it has been recognized that AMPK activation also has vascular effects. Vessels express AMPK‐α1 and ‐α2 isoforms; α1 predominates in endothelial cells and both are present in vascular smooth muscle with their relative predominance varying by tissue type (Evans et al. 2005; Goirand et al. 2007; Matsumoto et al. 2008). AMPK activation has been shown to improve cardiac function in a rat model of chronic heart failure (Wang et al. 2011); to help initiate hypoxic pulmonary vasoconstriction (Evans et al. 2005); and to augment acetylcholine‐induced relaxation in pre‐constricted isolated thoracic aorta, mesenteric or resistance‐sized cremaster arteries (Goirand et al. 2007; Ford & Rush, 2011). Importantly, the vascular effects of AMPK activation are not dependent on its metabolic effects (Bradley et al. 2010) or, as is the case with AICAR, the accumulation of adenosine (Evans et al. 2005; Goirand et al. 2007; Bradley et al. 2010), but rather are due to both NO‐dependent and NO‐independent mechanisms, namely, increased eNOS activity and NO production (Viollet et al. 2010), decreased superoxide and vasoconstrictor prostanoid production (Matsumoto et al. 2008; Li et al. 2010), and direct actions in vascular smooth muscle (Goirand et al. 2007).

Our data showing AMPK immunostaining in normoxic mouse uterine vessels and placenta tissue, and mRNA in murine and human placenta for both AMPK α isoforms and the upstream kinase responsible for activating AMPK, LKB1, in mouse and human placenta were consistent with prior reports showing that AMPK is expressed in placental tissue (Yung et al. 2012). This is, however, to the best of our knowledge the first report of its presence in murine uterine vessels during pregnancy. The UtA and other uterine vessels undergo profound changes during pregnancy, enlarging their diameters as the result of structural remodelling as well as increased vasodilator responses to flow, acetylcholine and other pharmacological agonists (reviewed in Osol & Moore, 2014). Such changes are attributable to effects of oestrogen and other pregnancy hormones serving to increase the production and/or activity of various vasodilators, including NO and large‐conductance potassium channels (BKCa), rather than placentation per se since the changes begin before placentation is complete, occur even in ectopic pregnancy, and are present albeit to a lesser degree in pseudo‐pregnant animals (Burchell, 1967; Rosenfeld et al. 1996; van der Heijden et al. 2005; Collins et al. 2011; Hu et al. 2011). The present report shows for the first time that uterine arteries are relaxed by AMPK activation, suggesting that activation of AMPK may be another mechanism contributing to pregnancy vasodilatation. The vasorelaxant effects of AMPK activation were due, in part, to increased NO production or activity as shown by the effect of NOS inhibition, but the major portion of its vasorelaxant effect remained following NOS inhibition, indicating that additional NO‐independent mechanisms are involved. One possibility is that AMPK activation reduced the production of vasoconstrictor prostanoids, similar to what has been reported in mesenteric arteries following metformin treatment in a rat model of type 2 diabetes (Matsumoto et al. 2008). Direct effects on vascular smooth muscle cells may also be involved via actions on the RhoA‐Rho associated protein kinase (Rock) pathway (Gayard et al. 2011) or via K+ channels given that AMPK activation affects multiple K+ channels (Andersen & Rasmussen, 2012), including the BKCa and the ATP‐sensitive potassium channel (KATP), previously implicated in uterine vascular responses to pregnancy and hypoxia (Zhu et al. 2013). Thus further study is required in both non‐pregnant and pregnant animals to determine whether pregnancy influences the effects of AMPK activation on UtA vasoreactivity, and the endothelial as well as vascular smooth muscle mechanisms involved.

Little is known about the effects of AMPK activation under conditions of hypoxia. Suggesting a beneficial role, Davidge and co‐workers have shown that resveratrol, which works in part by activating AMPK (Hardie, 2011; Tennen et al. 2012), administered to the mother under conditions of severe hypoxia dramatically improved fetal survival and increased placental relative to fetal weight but not fetal growth per se (Bourque et al. 2012; Banek et al. 2013). Resveratrol treatment also increased UtA blood flow velocity and fetal weight in a catechol‐O‐methyltransferase knockout mouse model of fetal growth restriction, but vasodilator or vasoconstrictor responses of isolated UtA were unaffected (Poudel et al. 2013). Resveratrol has also been shown to augment UtA blood flow during pregnancy in non‐human primates (Roberts et al. 2014).

We found that hypoxia in late murine pregnancy sufficient to decrease fetal growth increased the intensity of AMPK immunostaining in the labyrinthine zone, which may be more sensitive to hypoxia given its rich blood supply in contrast to the relatively hypovascularized junctional zone (Dilworth & Sibley, 2013). AMPK‐α2 mRNA expression was also greater in the labyrinthine than the junctional zone of the mouse placenta, suggesting that it may be the isoform involved in the greater immunostaining, but differences in primer efficiency prevent comparison of LKB1, AMPK‐α1 and ‐α2 expression levels. In humans, the levels of RT‐PCR products for both AMPK α‐subunits and LKB1 were greater in the placentas from high‐ vs. low‐altitude residents, suggesting higher basal AMPK activity and/or increased capacity to respond to metabolic stress. However, while these same placentas showed evidence of endoplasmic reticulum (ER) stress and mTOR inhibition, the ratio of P‐AMPK to total AMPK was not consistently elevated in the small number (n = 3) of non‐labouring placentas available for study (Yung et al. 2012). In pre‐eclamptic women, the ratio of P‐AMPK to total AMPK protein levels was inversely correlated with gestational age, being highest in those with the earliest onset of disease, although not higher overall in early‐ (<34 weeks) vs. late‐ (≥ 34 weeks) onset pre‐eclamptic or normotensive women (Yung et al. 2014 b). Thus, further studies in larger numbers of placentas are required to determine if AMPK is activated at high vs. low altitude in placental tissue.

We were interested in the effect of AMPK activation on UtA vasoreactivity under conditions of hypoxia given our prior studies indicating that the gene region containing PRKAA1 has been acted upon by natural selection in Andean residents of high altitude and that the variants more common in Andeans were positively associated with birth weight, UtA diameter and alterations in the expression of genes in the mTOR pathway previously implicated in altitude‐associated fetal growth restriction (Bigham et al. 2009; Yung et al. 2012; Bigham et al. 2014). The increased intensity of immunostaining in the mouse uterine arteries suggested that hypoxia increased AMPK abundance. However, since the antibody employed detects both α1 and α2 isoforms, we were not able to determine which isoforms were present in the uterine vessels.

Based on previous studies in pulmonary and uterine arteries, we expected that AMPK activation would have less vasodilator effect in the UtA isolated from the hypoxic vs. normoxic animals. In pulmonary arteries, AMPK activation under conditions of acute (≤60 min) hypoxia prompted vasoconstriction through mobilization of myocyte sarcoplasmic reticulum calcium stores via ryanodine receptors and the release of an endothelium‐derived constrictor (Evans, 2006). Inhibitory effects of AMPK activation on BKCa channels have also been observed, and in a manner that is splice‐variant specific (Wyatt et al. 2007; Ross et al. 2011). It is notable, therefore, that BKCa channels are inhibited in resistance‐sized ovine uterine vessels and, in turn, reverse the normal pregnancy reduction in myogenic tone (Hu et al. 2012). Further, we have reported that hypoxia throughout pregnancy reduced the NO‐dependent UtA vasodilator response to ACh and flow in isolated guinea pig UtA (White et al. 2000; Mateev et al. 2003). Therefore, our finding that pharmacological AMPK activation prompted similar concentration‐dependent vasodilatation in preconstricted UtA from normoxic and hypoxic animals was somewhat surprising. Since maximal contraction to PE tended (P = 0.07) to be greater in the hypoxic than the normoxic group and in order to evaluate the relationship between AMPK activation and PE constriction more fully, we extended our study to examine the effect of AMPK activation on contractile sensitivity to PE. We found that AMPK activation markedly reduced UtA contractile sensitivity to PE in the UtA from the hypoxic compared with normoxic animals as demonstrated by a rightward shift in the contractile‐response curves. The shift was greater in the vessels from the hypoxic compared to normoxic animals, and was partly reversed by treatment with the NOS inhibitor l‐NAME, suggesting a role for NO in opposing PE‐induced constriction. While we cannot rule out the possibility that hypoxic exposure increased NO sensitivity, this appeared unlikely given that the relaxation in response to ACh or A769662 was not greater in the UtA from the hypoxic compared to normoxic animals. In addition, even though the NO component was larger in the hypoxic group, the major portion of the greater reduction in contractile sensitivity to PE was NO independent. We therefore concluded that greater AMPK activation under conditions of hypoxic pregnancy opposes PE‐induced vasoconstriction, thus perhaps serving as a compensatory mechanism for maintaining uterine artery blood flow. Further studies are required for determining the mechanisms by which AMPK activation opposes PE‐induced vasoconstriction and whether similar effects of AMPK activation are observed when hypoxia is present throughout gestation such as is the case in residents of high altitude.

The strengths of our study were its broad‐ranging design in which human as well as murine tissues were examined following exposure to normoxia and either shorter‐ or longer‐term hypoxia. Further, multiple methods were used to evaluate the effects of AMPK activation and hypoxia, namely, immunohistochemistry, quantitative RT‐PCR and myography. Our results, however, were limited by use of a single AMPK activator. While A769662, unlike other agonists, activates AMPK directly (Goransson et al. 2007), it too has indirect effects by, for example, inhibiting the sodium pump (Benziane et al. 2009). While we used only one agonist, our results were similar to those obtained using metformin, AICAR or exercise to activate AMPK, and thus suggested that the responses observed were the result of AMPK activation and not some other effect of the agonist employed (Goirand et al. 2007; Ford & Rush, 2011; Wang et al. 2011; Kroller‐Schon et al. 2012), but additional studies using other agonists are required to confirm the mechanisms of A769662 action. Further studies are also needed using Western blot to document AMPK activation, knock‐out mice for clarifying the roles of the AMPK‐α1 and AMPK‐α2 isoforms, and pair‐fed animals to control for the lesser maternal weight gain seen during the period of hypoxic exposure in the present report. The possible effect of duration of hypoxic exposure also requires evaluation since the late‐pregnancy onset employed here does not parallel the circumstances of residence at high altitude where hypoxia is present throughout gestation but, on the other hand, it does likely resemble that occurring with pre‐eclampsia.

In summary, AMPK is present in maternal uterine vessels as well as in placental tissues, and its abundance increased by short‐as well as longer‐term hypoxia as indicated by greater immunostaining in uterine vessels from pregnant mice and increased expression of both catalytic AMPK subunits in murine and human placenta. This is, to our knowledge, the first report that pharmacological AMPK activation prompts vasodilatation in UtA via both NO‐dependent and NO‐independent mechanisms, and that exposure to chronic hypoxia sufficient to restrict fetal growth markedly augments the ability of AMPK activation to oppose PE‐induced vasoconstriction. Together with prior reports that genetic variation near AMPK is related to increased UtA blood flow and maintenance of fetal growth in high‐altitude adapted Andeans, we speculate that AMPK activation may be helping to maintain utero‐placental blood flow and thereby limit cellular energy depletion. In addition to its role as a metabolic sensor, AMPK may be a key link between maternal metabolic and cardiovascular responses to pregnancy and the regulation of fetal growth.

Additional information

Competing interests

None of the authors have any disclosures.

Author contributions

All authors contributed to the conception and design or analysis and interpretation of data, and the drafting of the article or revising it critically for important intellectual content. All authors provided final approval of the version to be published. In particular, the idea for the study originated with L.G.M. and G.J.B., A.L.F. and A.N.S.‐P. provided the animals, J.S.H. and A.N.S.‐P. collected the tissue, K.L.S. and D.A.G. carried out the myography studies, A.D.M. and A.M.E. conducted the quantitative RT‐PCR assays, and H.W.Y. and L.G.M. performed the immunohistochemistry. Studies were conducted at the Centre for Trophoblast Research and Department of Physiology, Development and Neuroscience at the University of Cambridge, Cambridge UK with the quantitative RT‐PCR assays being carried out at the Centre of Integrative Physiology at the University of Edinburgh, Edinburgh UK.

Funding

Funding for these studies was provided by the Wellcome Trust (084804/2/08/Z) to G.J.B., the British Heart Foundation and the Wellcome Trust to D.A.G., the Biotechnology and Biological Sciences Research Council (BBSRC) to A.L.F., a UK Wellcome Trust Programme Grant (WT081195MA) to A.M.E. and A.D.M., a BBSRC studentship and in vivo skills award to J.S.H., a National Health Medical Research Council and Centre for Trophoblast Research fellowship to A.N.S.‐P., and a NIH RO1 grant (HLBI‐079647) to L.G.M. along with sabbatical support from Wake Forest University.

Acknowledgements

We thank Melanie Monk and Dr Lori Walker for their help with the immunostaining and its quantification. We also thank the staff of the animal facility for the care of the animals, and Dr Martha Tissot van Patot and the health‐care providers for their help in collecting human placentas at the sea‐level and high‐altitude sites.

References

  1. Alkorta‐Aranburu G, Beall CM, Witonsky DB, Gebremedhin A, Pritchard JK & Di Rienzo A (2012). The genetic architecture of adaptations to high altitude in Ethiopia. PLoS Genet 8, e1003110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Andersen MN & Rasmussen HB (2012). AMPK: A regulator of ion channels. Commun Integr Biol 5, 480–484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banek CT, Bauer AJ, Needham KM, Dreyer HC & Gilbert JS (2013). AICAR administration ameliorates hypertension and angiogenic imbalance in a model of preeclampsia in the rat. Am J Physiol Heart Circ Physiol 304, H1159–H1165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barcroft J (1933). The conditions of fetal respiration. Lancet 222, 1021–1024. [Google Scholar]
  5. Barron DH, Metcalfe J, Meschia G, Huckabee W, Hellegers A & Prystowsky H (1964). Adaptations of pregnant ewes and their fetuses to high altitude In The Physiological Effects of High Altitude, ed. Weihe WH, pp. 115–129. Macmillan, New York. [Google Scholar]
  6. Beall CM, Cavalleri GL, Deng L, Elston RC, Gao Y, Knight J, Li C, Li JC, Liang Y, McCormack M, Montgomery HE, Pan H, Robbins PA, Shianna KV, Tam SC, Tsering N, Veeramah KR, Wang W, Wangdui P, Weale ME, Xu Y, Xu Z, Yang L, Zaman MJ, Zeng C, Zhang L, Zhang X, Zhaxi P & Zheng YT (2010). Natural selection on EPAS1 (HIF2α) associated with low hemoglobin concentration in Tibetan highlanders. Proc Natl Acad Sci USA 107, 11459–11464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett A, Sain SR, Vargas E & Moore LG (2008). Evidence that parent‐of‐origin affects birth‐weight reductions at high altitude. Am J Human Biol 20, 592–597. [DOI] [PubMed] [Google Scholar]
  8. Benziane B, Bjornholm M, Lantier L, Viollet B, Zierath JR & Chibalin AV (2009). AMP‐activated protein kinase activator A‐769662 is an inhibitor of the Na+‐K+‐ATPase. Am J Physiol Cell Physiol 297, C1554–C1566. [DOI] [PubMed] [Google Scholar]
  9. Bigham A, Bauchet M, Pinto D, Mao X, Akey JM, Mei R, Scherer SW, Julian CG, Wilson MJ, Lopez Herraez D, Brutsaert T, Parra EJ, Moore LG & Shriver MD (2010). Identifying signatures of natural selection in Tibetan and Andean populations using dense genome scan data. PLoS Genet 6, e1001116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bigham AW, Julian CG, Wilson MJ, Vargas E, Browne VA, Shriver MD & Moore LG (2014). Maternal PRKAA1 and EDNRA genotypes are associated with birth weight, and PRKAA1 with uterine artery diameter and metabolic homeostasis at high altitude. Physiol Genomics 46, 687–697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bigham AW, Mao X, Mei R, Brutsaert T, Wilson MJ, Julian CG, Parra EJ, Akey JM, Moore LG & Shriver MD (2009). Identifying positive selection candidate loci for high‐altitude adaptation in Andean populations. Hum Genomics 4, 79–90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bourque SL, Dolinsky VW, Dyck JR & Davidge ST (2012). Maternal resveratrol treatment during pregnancy improves adverse fetal outcomes in a rat model of severe hypoxia. Placenta 33, 449–452. [DOI] [PubMed] [Google Scholar]
  13. Bradley EA, Eringa EC, Stehouwer CD, Korstjens I, van Nieuw Amerongen GP, Musters R, Sipkema P, Clark MG & Rattigan S (2010). Activation of AMP‐activated protein kinase by 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside in the muscle microcirculation increases nitric oxide synthesis and microvascular perfusion. Arterioscler Thromb Vasc Biol 30, 1137–1142. [DOI] [PubMed] [Google Scholar]
  14. Burchell RC (1967). Arterial blood flow into the human intervillous space. Am J Obstet Gynecol 98, 303–311. [DOI] [PubMed] [Google Scholar]
  15. Chen D, Zhou X, Zhu Y, Zhu T & Wang J (2002). Comparison study on uterine and umbilical artery blood flow during pregnancy at high altitude and at low altitude. Zhonghua Fu Chan Ke Za Zhi 37, 69–71. [PubMed] [Google Scholar]
  16. Collins SL, Grant D, Black RS, Vellayan M & Impey L (2011). Abdominal pregnancy: a perfusion confusion? Placenta 32, 793–795. [DOI] [PubMed] [Google Scholar]
  17. Dilworth MR & Sibley CP (2013). Review: Transport across the placenta of mice and women. Placenta 34 Suppl, S34–S39. [DOI] [PubMed] [Google Scholar]
  18. Evans AM (2006). AMP‐activated protein kinase and the regulation of Ca2+ signalling in O2‐sensing cells. J Physiol 574, 113–123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Evans AM, Hardie DG, Peers C, Wyatt CN, Viollet B, Kumar P, Dallas ML, Ross F, Ikematsu N, Jordan HL, Barr BL, Rafferty JN & Ogunbayo O (2009). Ion channel regulation by AMPK: the route of hypoxia‐response coupling in the carotid body and pulmonary artery. Ann N Y Acad Sci 1177, 89–100. [DOI] [PubMed] [Google Scholar]
  20. Evans AM, Mustard KJ, Wyatt CN, Peers C, Dipp M, Kumar P, Kinnear NP & Hardie DG (2005). Does AMP‐activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2‐sensing cells? J Biol Chem 280, 41504–41511. [DOI] [PubMed] [Google Scholar]
  21. Ford RJ & Rush JW (2011). Endothelium‐dependent vasorelaxation to the AMPK activator AICAR is enhanced in aorta from hypertensive rats and is NO and EDCF dependent. Am J Physiol Heart Circ Physiol 300, H64–H75. [DOI] [PubMed] [Google Scholar]
  22. Gant N & Worley R (1989). Measurement of uteroplacental blood flow in the human In The Uterine Circulation, ed. Rosenfeld CR. Perinatology Press, Ithaca, NY, 53–70. [Google Scholar]
  23. Gayard M, Guilluy C, Rousselle A, Viollet B, Henrion D, Pacaud P, Loirand G & Rolli‐Derkinderen M (2011). AMPK alpha 1‐induced RhoA phosphorylation mediates vasoprotective effect of estradiol. Arterioscler Thromb Vasc Biol 31, 2634–2642. [DOI] [PubMed] [Google Scholar]
  24. Giussani DA, Camm EJ, Niu Y, Richter HG, Blanco CE, Gottschalk R, Blake EZ, Horder KA, Thakor AS, Hansell JA, Kane AD, Wooding FB, Cross CM & Herrera EA (2012). Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress. PloS One 7, e31017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Giussani DA, Phillips PS, Anstee S & Barker DJ (2001). Effects of altitude versus economic status on birth weight and body shape at birth. Pediatr Res 49, 490–494. [DOI] [PubMed] [Google Scholar]
  26. Goirand F, Solar M, Athea Y, Viollet B, Mateo P, Fortin D, Leclerc J, Hoerter J, Ventura‐Clapier R & Garnier A (2007). Activation of AMP kinase α1 subunit induces aortic vasorelaxation in mice. J Physiol 581, 1163–1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Goransson O, McBride A, Hawley SA, Ross FA, Shpiro N, Foretz M, Viollet B, Hardie DG & Sakamoto K (2007). Mechanism of action of A‐769662, a valuable tool for activation of AMP‐activated protein kinase. J Biol Chem 282, 32549–32560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hardie DG (2011). Sensing of energy and nutrients by AMP‐activated protein kinase. Am J Clin Nutr 93, 891S–896S. [DOI] [PubMed] [Google Scholar]
  29. Hardie DG, Ross FA & Hawley SA (2012). AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol 13, 251–262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hu XQ, Xiao D, Zhu R, Huang X, Yang S, Wilson S & Zhang L (2011). Pregnancy upregulates large‐conductance Ca2+‐activated K+ channel activity and attenuates myogenic tone in uterine arteries. Hypertension 58, 1132–1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Hu XQ, Xiao D, Zhu R, Huang X, Yang S, Wilson SM & Zhang L (2012). Chronic hypoxia suppresses pregnancy‐induced upregulation of large‐conductance Ca2+‐activated K+ channel activity in uterine arteries. Hypertension 60, 214–222. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Julian CG, Galan HL, Wilson MJ, Desilva W, Cioffi‐Ragan D, Schwartz J & Moore LG (2008). 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 295, R906–R915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Julian CG, Hageman JL, Wilson MJ, Vargas E & Moore LG (2011). Lowland origin women raised at high altitude are not protected against lower uteroplacental O2 delivery during pregnancy or reduced birth weight. Am J Human Biol 23, 509–516. [DOI] [PubMed] [Google Scholar]
  34. Julian CG, Vargas E, Armaza JF, Wilson MJ, Niermeyer S & Moore LG (2007). High‐altitude ancestry protects against hypoxia‐associated reductions in fetal growth. Arch Dis Child Fetal Neonatal Ed 92, F372–E377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Julian CG, Wilson MJ, Lopez M, Yamashiro H, Tellez W, Rodriguez A, Bigham AW, Shriver MD, Rodriguez C, Vargas E & Moore LG (2009). Augmented uterine artery blood flow and oxygen delivery protect Andeans from altitude‐associated reductions in fetal growth. Am J Physiol Regul Integr Comp Physiol 296, R1564–R1575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kroller‐Schon S, Jansen T, Hauptmann F, Schuler A, Heeren T, Hausding M, Oelze M, Viollet B, Keaney JF Jr, Wenzel P, Daiber A, Munzel T & Schulz E (2012). α1AMP‐activated protein kinase mediates vascular protective effects of exercise. Arterioscler Thromb Vasc Biol 32, 1632–1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Li D, Zhang Y, Ma J, Ling W & Xia M (2010). Adenosine monophosphate activated protein kinase regulates ABCG1‐mediated oxysterol efflux from endothelial cells and protects against hypercholesterolemia‐induced endothelial dysfunction. Arterioscler Thromb Vasc Biol 30, 1354–1362. [DOI] [PubMed] [Google Scholar]
  38. Mateev S, Sillau AH, Mouser R, McCullough RE, White MM, Young DA & Moore LG (2003). Chronic hypoxia opposes pregnancy‐induced increase in uterine artery vasodilator response to flow. Am J Physiol Heart Circ Physiol 284, H820–H829. [DOI] [PubMed] [Google Scholar]
  39. Matsumoto T, Noguchi E, Ishida K, Kobayashi T, Yamada N & Kamata K (2008). Metformin normalizes endothelial function by suppressing vasoconstrictor prostanoids in mesenteric arteries from OLETF rats, a model of type 2 diabetes. Am J Physiol Heart Circ Physiol 295, H1165–H1176. [DOI] [PubMed] [Google Scholar]
  40. Moore LG, Niermeyer S & Zamudio S (1998). Human adaptation to high altitude: regional and life‐cycle perspectives. Am J Phys Anthropol 27 Suppl, 25–64. [DOI] [PubMed] [Google Scholar]
  41. Moore LG, Zamudio S, Zhuang J, Sun S & Droma T (2001). Oxygen transport in Tibetan women during pregnancy at 3658 m. Am J Phys Anthropol 114, 42–53. [DOI] [PubMed] [Google Scholar]
  42. Osol G & Moore LG (2014). Maternal uterine vascular remodeling during pregnancy. Microcirculation 21, 38–47. [DOI] [PubMed] [Google Scholar]
  43. Poudel R, Stanley JL, Rueda‐Clausen CF, Andersson IJ, Sibley CP, Davidge ST & Baker PN (2013). Effects of resveratrol in pregnancy using murine models with reduced blood supply to the uterus. PloS One 8, e64401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pulgar VM, Yamashiro H, Rose JC & Moore LG (2011). Role of the AT2 receptor in modulating the angiotensin II contractile response of the uterine artery at mid‐gestation. J Renin Angiotensin Aldosterone Syst 12, 176–183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Roberts VH, Pound LD, Thorn SR, Gillingham MB, Thornburg KL, Friedman JE, Frias AE & Grove KL (2014). Beneficial and cautionary outcomes of resveratrol supplementation in pregnant nonhuman primates. FASEB J 28, 2466–2477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rosenfeld CR, Cox BE, Roy T & Magness RR (1996). Nitric oxide contributes to estrogen‐induced vasodilation of the ovine uterine circulation. J Clin Invest 98, 2158–2166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ross FA, Rafferty JN, Dallas ML, Ogunbayo O, Ikematsu N, McClafferty H, Tian L, Widmer H, Rowe IC, Wyatt CN, Shipston MJ, Peers C, Hardie DG & Evans AM (2011). Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium‐ and voltage‐activated potassium channel confers regulation by AMP‐activated protein kinase. J Biol Chem 286, 11929–11936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Simonson TS, Yang Y, Huff CD, Yun H, Qin G, Witherspoon DJ, Bai Z, Lorenzo FR, Xing J, Jorde LB, Prchal JT & Ge R (2010). Genetic evidence for high‐altitude adaptation in Tibet. Science 329, 72–75. [DOI] [PubMed] [Google Scholar]
  49. Soria R, Julian C, Vargas E, Moore L & Giussani D (2013). Graduated effects of high‐altitude hypoxia and highland ancestry on birth size. Pediatr Res 74, 633–638. [DOI] [PubMed] [Google Scholar]
  50. Tennen RI, Michishita‐Kioi E & Chua KF (2012). Finding a target for resveratrol. Cell 148, 387–389. [DOI] [PubMed] [Google Scholar]
  51. van der Heijden OW, Essers YP, Spaanderman ME, De Mey JG, van Eys GJ & Peeters LL (2005). Uterine artery remodeling in pseudopregnancy is comparable to that in early pregnancy. Biol Reprod 73, 1289–1293. [DOI] [PubMed] [Google Scholar]
  52. Viollet B, Athea Y, Mounier R, Guigas B, Zarrinpashneh E, Horman S, Lantier L, Hebrard S, Devin‐Leclerc J, Beauloye C, Foretz M, Andreelli F, Ventura‐Clapier R & Bertrand L (2009). AMPK: Lessons from transgenic and knockout animals. Front Biosci 14, 19–44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Viollet B, Guigas B, Sanz Garcia N, Leclerc J, Foretz M & Andreelli F (2012). Cellular and molecular mechanisms of metformin: an overview. Clin Sci (Lond) 122, 253–270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Viollet B, Horman S, Leclerc J, Lantier L, Foretz M, Billaud M, Giri S & Andreelli F (2010). AMPK inhibition in health and disease. Crit Rev Biochem Mol Biol 45, 276–295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wang X & Proud CG (2006). The mTOR pathway in the control of protein synthesis. Physiology (Bethesda) 21, 362–369. [DOI] [PubMed] [Google Scholar]
  56. Wang XF, Zhang JY, Li L, Zhao XY, Tao HL & Zhang L (2011). Metformin improves cardiac function in rats via activation of AMP‐activated protein kinase. Clin Exp Pharmacol Physiol 38, 94–101. [DOI] [PubMed] [Google Scholar]
  57. White MM, McCullough RE, Dyckes R, Robertson AD & Moore LG (2000). Chronic hypoxia, pregnancy, and endothelium‐mediated relaxation in guinea pig uterine and thoracic arteries. Am J Physiol Heart Circ Physiol 278, H2069–H2075. [DOI] [PubMed] [Google Scholar]
  58. Wyatt CN, Mustard KJ, Pearson SA, Dallas ML, Atkinson L, Kumar P, Peers C, Hardie DG & Evans AM (2007). AMP‐activated protein kinase mediates carotid body excitation by hypoxia. J Biol Chem 282, 8092–8098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Xiao D, Longo LD & Zhang L (2010). 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 203, 596e6–596e12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yi X, Liang Y, Huerta‐Sanchez E, Jin X, Cuo ZX, Pool JE, Xu X, Jiang H, Vinckenbosch N, Korneliussen TS, Zheng H, Liu T, He W, Li K, Luo R, Nie X, Wu H, Zhao M, Cao H, Zou J, Shan Y, Li S, Yang Q, Asan, Ni P, Tian G, Xu J, Liu X, Jiang T, Wu R, Zhou G, Tang M, Qin J, Wang T, Feng S, Li G, Huasang, Luosang J, Wang W, Chen F, Wang Y, Zheng X, Li Z, Bianba Z, Yang G, Wang X, Tang S, Gao G, Chen Y, Luo Z, Gusang L, Cao Z, Zhang Q, Ouyang W, Ren X, Liang H, Huang Y, Li J, Bolund L, Kristiansen K, Li Y, Zhang Y, Zhang X, Li R, Yang H, Nielsen R & Wang J (2010). Sequencing of 50 human exomes reveals adaptation to high altitude. Science 329, 75–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yung HW, Atkinson D, Campion‐Smith T, Olovsson M, Charnock‐Jones DS & Burton GJ (2014. a). Differential activation of placental unfolded protein response pathways implies heterogeneity in causation of early‐ and late‐onset pre‐eclampsia. J Pathol 234, 262–276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Yung HW, Colleoni F, Atkinson D, Cook E, Murray AJ, Burton GJ & Charnock‐Jones DS (2014. b). Influence of speed of sample processing on placental energetics and signalling pathways: implications for tissue collection. Placenta 35, 103–108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Yung HW, Cox M, Tissot van Patot M & Burton GJ (2012). Evidence of endoplasmic reticulum stress and protein synthesis inhibition in the placenta of non‐native women at high altitude. FASEB J 26, 1970–1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zamudio S, Palmer SK, Droma T, Stamm E, Coffin C & Moore LG (1995). Effect of altitude on uterine artery blood flow during normal pregnancy. J Appl Physiol 79, 7–14. [DOI] [PubMed] [Google Scholar]
  65. Zhu R, Xiao D & Zhang L (2013). Potassium channels and uterine vascular adaptation to pregnancy and chronic hypoxia. Curr Vasc Pharmacol 11, 737–747. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Physiology are provided here courtesy of The Physiological Society

RESOURCES