AMPK function in uterine vasculature and placenta at high altitude

Abstract

The chronic hypoxia of high altitude (HA, >2500m) residence reduces uterine artery (UtA) blood flow, contributing to an increased frequency of foetal growth restriction (FGR). FGR pregnancies have reduced UtA blood flow due partially to impaired myometrial artery (MyoA) vasodilation. However, studies show lower rates of HA-associated reductions in foetal growth in Andeans and describe an association between genetic variants predicted to activate the AMP-activated protein kinase (AMPK) pathway and protection against low birth weight. Vascular function studies show that AMPK-dependent vasodilator responses are amplified in UtA from mice exposed to hypoxia during pregnancy and MyoA from pregnant women at HA, while the response is reduced in MyoA from women with FGR pregnancies. The effect of HA (or gestational hypoxia) on placental AMPK activation needs to be clarified, with some studies showing an effect and others not. There is potential to use AMPK activators as therapeutic targets. However, some drugs (i.e., metformin) approved for use in pregnancy complications cause off-target and adverse metabolic effects in offspring, which discourage their use. Future studies are warranted to elucidate the mechanisms underlying the altitude-dependent activation of AMPK in uncomplicated pregnancies and its reduction in FGR to identify possible vascular-specific targets for therapeutic intervention.

High-altitude pregnancies

Over 80 million people reside at high altitude (HA), defined as >2500 m above sea level (1). HA residence has detrimental effects on pregnancy, increasing the risk of developing vascular disorders of pregnancy, such as foetal growth restriction (FGR) and preeclampsia (2–4). This is likely due to blunting of the regular pregnancy-dependent vasodilation of the uterine vasculature in human pregnancies at HA (5), and has also been observed in animal models exposed to hypoxic conditions during pregnancy (6–8). This reduction could be associated with a diminished pregnancy-dependent rise in uterine artery (UtA) blood flow, leading to reduced foetal growth (3, 9). However, not all infants born at HA show reduced birth weight (10).

Compared to newcomers, multigenerational HA populations are relatively protected from the adverse effects of HA residency on UtA blood flow and foetal growth (11–15). A selected-for single nucleotide polymorphism (SNP) in the PRKAA1 gene, which encodes for the AMP-activated protein kinase (AMPK), has been associated with higher UtA blood flow and greater birth weight in HA populations of Andean origin (16), suggesting a mechanism for HA adaptation.

AMPK effect on uterine vasculature at HA or under hypoxic conditions.

AMPK is a metabolic sensor activated by hypoxia. AMPK regulates many cellular processes, such as glucose homeostasis, fatty acid oxidation and synthesis, protein synthesis, and autophagy, among others (17). Several mechanisms have been proposed to underlie the hypoxia-dependent activation of AMPK (reviewed by (18)): the canonical AMPK activation via increased AMP/ATP ratio is a likely mechanism, as lower oxygen levels impair oxidative phosphorylation, decreasing mitochondrial ATP production, causing activation of liver kinase B1 (LKB1), which directly phosphorylates AMPK, promoting its activation (19, 20). Another possible mechanism of AMPK activation by hypoxia is through increased Ca²⁺ levels and Ca²⁺/calmodulin-dependent protein kinase 2 (CaMKK2) (21, 22), an upstream activator of AMPK. Furthermore, impaired mitochondrial function (such as that observed during hypoxia) could also promote the accumulation of reactive oxygen species (ROS) (23), which, in turn, may activate AMPK (24, 25), although others have found no effect of ROS scavengers in the hypoxia-dependent activation of AMPK (19). These dissimilar observations suggest that these mechanisms are tissue-specific, as either LKB1, CaMKK2, or ROS could independently activate AMPK in response to hypoxia, depending on the cell type studied (18).

In the vasculature, AMPK activation induces vasodilation (26–28). Multiple mechanisms have been implicated in AMPK-dependent vasodilation, such as increased activation of endothelial nitric oxide (NO) synthase (eNOS) (27, 28) and activation of large-conductance Ca²⁺-activated K⁺ channels via Ca²⁺ release from intracellular Ca²⁺ stores in the smooth muscle cells (29). Therefore, this pathway may have important implications for pregnancy. Notably, mice exposed to hypoxia during pregnancy show more AMPK-mediated vasodilation of the UtA than animals kept under normoxic conditions (30). Furthermore, in vivo treatment with an AMPK activator, AICAR, partially prevents the reduction in foetal growth induced by hypoxia in a murine model by increasing UtA blood flow (31). Notably, in vivo AMPK activation only increased the proportion of cardiac output flowing through the UtA in hypoxic mice, indicating that it may selectively affect the uteroplacental circulation under hypoxic conditions (31). Human myometrial arteries (MyoA) – also known as radial arteries – are critical regulators of uteroplacental blood flow (32). In uncomplicated, appropriate-for-gestational-age (AGA) pregnancies from women residing at HA, AMPK activation has a larger vasodilatory effect on the MyoA than women living at lower altitudes (~1700 m) (33). Conversely, pregnancies affected with FGR at HA show reduced AMPK-dependent vasodilation compared to AGA pregnant women (33), suggesting a role of AMPK in maintaining the vasodilatory capacity of MyoA at HA under conditions to preserve foetal growth. The mechanism by which AMPK activation elicits vasodilation in the MyoA depends on the altitudinal residence. For instance, in AGA pregnancies at low altitudes, the AMPK-evoked MyoA vasodilation is mainly mediated by smooth muscle signalling. In contrast, in AGA pregnancies at HA, eNOS plays a role in the MyoA vasodilation induced by AMPK (33). As described above, several mechanisms have been shown to link hypoxia with AMPK activation (18). However, the mechanisms underlying the hypoxia-dependent activation of AMPK in the uteroplacental circulation remain unknown.

Thus, as summarized in Figure 1, in low-altitude AGA pregnancies, the role of AMPK in MyoA vasodilation is minor and mainly restricted to smooth muscle signalling; other vasodilators such as acetylcholine and bradykinin may be primarily responsible for the increased pregnancy-mediated MyoA vasodilation (5). Meanwhile, at HA, endothelial AMPK-dependent MyoA vasodilation, via NO signalling, becomes more critical in maintaining foetal growth (33). Furthermore, a particular AMPK SNP is associated with higher UtA diameter (blood flow) and birth weight in Andeans (16). However, the functional role of this SNP in MyoA vasodilation remains unknown. Conversely, HA pregnancies with FGR present a reduced AMPK- and acetylcholine-dependent vasodilation (33) and reduced UtA diameter and blood flow (3).

Figure 1. Effect of high altitude on AMPK signaling in the uterine circulation.

See text for description. Green plus (+) and red minus (−) symbols represent increased or reduced vasodilatory responses to each factor, respectively. Black question (?) symbols represent data not available. Abbreviations (in alphabetical order): ACh, acetylcholine; AGA, appropriate for gestation age; AMPK, AMP-activated protein kinase; BK, bradykinin; eNOS, endothelial nitric oxide synthase; FGR, fetal growth restriction; P, phosphorylation; SNPs, single nucleotide polymorphisms); UtA, uterine artery.

Placental AMPK in HA pregnancies

AMPK is a master regulator of several metabolic pathways, integrating hypoxic stimuli with metabolism. However, AMPK’s role in placental function, a highly metabolic organ, is not clear. The effect of HA, or hypoxia, on placental AMPK activation during pregnancy is also controversial. A study of human placentas from sea-level and HA residents (~3100 m) showed no difference in the activation of AMPK, measured as the ratio of phosphorylated AMPKα to total AMPKα (34). Another study found increased mRNA expression of AMPKα in whole placentas from HA compared to low-altitude women (30). In a more recent report, the ratio of phosphorylated-to-total AMPKα protein expression was increased in placentas from HA and moderate altitude (~1700 m) compared to sea level (35). There was also an altitude-dependent increment in the expression of the phosphorylated form of the tuberous sclerosis complex 2 (TSC2) (35), a downstream target of AMPK, confirming the increased activity of AMPK in these placentas. The apparent discrepancies between these studies could be due to the variability among different human populations, the gradient of altitudes compared between studies, or differences in the collection and processing of the samples.

Similarly, reports in mice exposed to hypoxia during pregnancy have shown contradicting results. One study found that hypoxia-exposed mice showed increased expression of AMPKα in the placental labyrinth zone, where the exchange between the mother and foetus occurs (30). In contrast, another report showed that the phosphorylation of AMPKα is reduced in the placenta of animals exposed to hypoxia during late pregnancy (36). These differences could be attributable to experimental differences, such as the modality of hypoxic exposure (i.e., hypoxic hypoxia vs. hypobaric hypoxia) or the time of exposure during pregnancy (i.e., days of gestation 14–19 vs. 14.5–18.5). Future functional cellular and physiological studies may help to elucidate the role of AMPK in the regulation of placenta metabolism and placental vascular function.

Maternal AMPK gene expression in HA

Genotypic studies comparing multigenerational Andean and historically low-altitude-dwelling European populations have shown a positive selection for PRKAA1 in Andeans that is associated with relative protection from HA effects on UtA diameter at week 36 of gestation and birth weight (16). A recent report studied maternal expression of PRKAA1 and related genes (in peripheral blood mononuclear cells) in populations from low altitude and HA in Colorado at two gestational ages (20 and 34 weeks). Although PRKAA1 expression did not change with altitude or gestational age, there was a correlation between PRKAA1 expression and UtA diameter and blood flow at 20 weeks of gestation in the HA group and between PRKAA1 expression and UtA diameter at 34 weeks of gestation in the low-altitude group (37). These observations are consistent with those in Andean populations. However, further studies should perform genotypic analyses in non-Andean women with uncomplicated pregnancies at HA and functional vascular studies in Andean populations to dissect the underlying ‘protective’ mechanism(s) of AMPK signalling in the vasculature during pregnancy.

Discussion and future directions

The natural laboratory of residence at HA offers valuable insights into the human vascular adaptation to pregnancy under conditions of chronic hypoxia. Hence, studies at HA can provide a deeper understanding of the pathophysiology of vascular-associated pregnancy complications such as FGR and preeclampsia.

The role of AMPK in the adaptive responses to HA during pregnancy, particularly in the uterine vasculature, makes it a strong candidate for therapeutic intervention. Drugs that activate AMPK (i.e., metformin) represent an interesting possibility. The use of metformin has been approved for the treatment of complications of pregnancy, such as gestational diabetes mellitus or polycystic ovary syndrome (38). However, clinical randomized controlled trials indicate that in utero exposure to metformin may lead to metabolic issues, increasing the risk of obesity in children (39, 40). Possible off-target effects are also likely since metformin can cross the placenta and reach the foetus (41, 42). Unfortunately, the broad effects of metformin discourage its use for selectively increasing uterine vasodilation in cases of reduced uteroplacental perfusion. For this reason, identifying vascular-specific targets of AMPK, such as K⁺ channels and others, may offer a more selective way of stimulating uterine vasodilation in cases of vascular disorders of pregnancy.