Gestational Hypoxia Research Articles

Long-term high altitude hypoxia during gestation suppresses large conductance Ca2+ -activated K+ channel function in uterine arteries: a causal role for microRNA-210.

Gestational hypoxia represses ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which is recovered by inhibiting endogenous miR-210. Inhibition of miR-210 rescues BKCa channel expression and current in uterine arteries of pregnant animals acclimatized to high altitude hypoxia in a TET-dependent manner. miR-210 blockade restores BKCa channel-mediated relaxations and attenuates pressure-dependent myogenic tone in uterine arteries of pregnant animals acclimatized to high altitude. Gestational hypoxia at high altitude has profound adverse effects on the uteroplacental circulation, and is associated with increased incidence of preeclampsia and fetal intrauterine growth restriction. Previous studies demonstrated that suppression of large-conductance Ca2+ -activated K+ (BKCa ) channel function played a critical role in the maladaptation of uteroplacental circulation caused by gestational hypoxia. Yet, the mechanisms underlying gestational hypoxia-induced BKCa channel repression remain undetermined. The present study investigated a causal role of microRNA-210 (miR-210) in hypoxia-mediated repression of BKCa channel expression and function in uterine arteries using a sheep model. The results revealed that gestational hypoxia significantly decreased ten-eleven translocation methylcytosine dioxygenase 1 (TET1) expression in uterine arteries, which was recovered by inhibiting endogenous miR-210 with miR-210 locked nucleic acid (miR-210-LNA). Of importance, miR-210-LNA restored BKCa channel β1 subunit expression in uterine arteries, which was blocked by a competitive TET inhibitor, fumarate, thus functionally linking miR-210 to the TET1-BKCa channel cascade. In addition, miR-210-LNA reversed hypoxia-mediated suppression of BKCa channel function and rescued the effect of steroid hormones in upregulating BKCa channel expression and function in uterine arteries, which were also ablated by fumarate. Collectively, the present study demonstrates a causative effect of miR-210 in the downregulation of TET1 and subsequent repression of BKCa channel expression and function, providing a novel mechanistic insight into the regulation of BKCa channel function and the molecular basis underlying the maladaptation of uterine vascular function in gestational hypoxia.

Gestational Hypoxia and Developmental Plasticity.

Hypoxia is one of the most common and severe challenges to the maintenance of homeostasis. Oxygen sensing is a property of all tissues, and the response to hypoxia is multidimensional involving complicated intracellular networks concerned with the transduction of hypoxia-induced responses. Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. Hypoxia during gestation impacts both the mother and fetal development through interactions with an individual's genetic traits acquired over multiple generations by natural selection and changes in gene expression patterns by altering the epigenetic code. Changes in the epigenome determine "genomic plasticity," i.e., the ability of genes to be differentially expressed according to environmental cues. The genomic plasticity defined by epigenomic mechanisms including DNA methylation, histone modifications, and noncoding RNAs during development is the mechanistic substrate for phenotypic programming that determines physiological response and risk for healthy or deleterious outcomes. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue. The complex molecular and epigenetic interactions that may impact an individual's physiology and developmental programming of health and disease later in life are discussed.

Early Gestational Hypoxia and Adverse Developmental Outcomes.

Hypoxia is a normal and essential part of embryonic development. However, this state may leave the embryo vulnerable to damage when oxygen supply is disturbed. Embryofetal response to hypoxia is dependent on duration and depth of hypoxia, as well as developmental stage. Early postimplantation rat embryos were resilient to hypoxia, with many surviving up to 1.5 hr of uterine clamping, while most mid-gestation embryos were dead after 1 hour of clamping. Survivors were small and many had a range of defects, principally terminal transverse limb reduction defects. Similar patterns of malformations occurred when embryonic hypoxia was induced by maternal hypoxia, interruption of uteroplacental flow, or perfusion and embryonic bradycardia. There is good evidence that high altitude pregnancies are associated with smaller babies and increased risk of some malformations, but these results are complicated by increased risk of pre-eclampsia. Early onset pre-eclampsia itself is associated with small for dates and increased risk of atrio-ventricular septal defects. Limb defects have clearly been associated with chorionic villus sampling, cocaine, and misoprostol use. Similar defects are also observed with increased frequency among fetuses who are homozygous for thalassemia. Drugs that block the potassium current, whether as the prime site of action or as a side effect, are highly teratogenic in experimental animals. They induce embryonic bradycardia, hypoxia, hemorrhage, and blisters, leading to transverse limb defects as well as craniofacial and cardiovascular defects. While evidence linking these drugs to birth defects in humans is not compelling, the reason may methodological rather than biological. Birth Defects Research 109:1358-1376, 2017.© 2017 Wiley Periodicals, Inc.

Early gestational intermittent hypoxia in rats induces delayed changes in immune and redox homeostasis with lasting perivascular placental edema

Early gestational intermittent hypoxia in rats induces delayed changes in immune and redox homeostasis with lasting perivascular placental edema

Gestational hypoxia disrupts the neonatal leptin surge and programs hyperphagia and obesity in male offspring in the Sprague-Dawley rat.

The effect of gestational hypoxia on the neonatal leptin surge, development of hypothalamic arcuate nuclei (ARH) projections and appetite that could contribute to the programming of offspring obesity is lacking. We examined the effect of 12% O2 from gestational days 15–19 in the Sprague-Dawley rat on post-weaning appetite, fat deposition by MRI, adipose tissue cytokine expression, the neonatal leptin surge, ARH response to exogenous leptin, and αMSH projections to the paraventricular nucleus (PVN) in response to a high fat (HFD) or control diet (CD) in male offspring. Normoxia (NMX) and Hypoxia (HPX) offspring exhibited increased food intake when fed a HFD from 5–8 weeks post-birth; HPX offspring on the CD had increased food intake from weeks 5–7 vs. NMX offspring on a CD. HPX offspring on a HFD remained hyperphagic through 23 weeks. Body weight were the same between offspring from HPX vs. NMX dams from 4–12 weeks of age fed a CD or HFD. By 14–23 weeks of age, HPX offspring fed the CD or HFD as well as male NMX offspring fed the HFD were heavier vs. NMX offspring fed the CD. HPX offspring fed a CD exhibited increased abdominal adiposity (MRI) that was amplified by a HFD. HPX offspring fed a HFD exhibited the highest abdominal fat cytokine expression. HPX male offspring had higher plasma leptin from postnatal day (PN) 6 through 14 vs. NMX pups. HPX offspring exhibited increased basal c-Fos labeled cells in the ARH vs. NMX pups on PN16. Leptin increased c-Fos staining in the ARH in NMX but not HPX offspring at PN16. HPX offspring had fewer αMSH fibers in the PVN vs. NMX offspring on PN16. In conclusion, gestational hypoxia impacts the developing ARH resulting in hyperphagia contributing to adult obesity on a control diet and exacerbated by a HFD.

Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development

Some neuropsychiatric disease, including schizophrenia, may originate during prenatal development, following periods of gestational hypoxia and placental oxidative stress. Here we investigated if gestational hypoxia promotes damaging secretions from the placenta that affect fetal development and whether a mitochondria-targeted antioxidant MitoQ might prevent this. Gestational hypoxia caused low birth-weight and changes in young adult offspring brain, mimicking those in human neuropsychiatric disease. Exposure of cultured neurons to fetal plasma or to secretions from the placenta or from model trophoblast barriers that had been exposed to altered oxygenation caused similar morphological changes. The secretions and plasma contained altered microRNAs whose targets were linked with changes in gene expression in the fetal brain and with human schizophrenia loci. Molecular and morphological changes in vivo and in vitro were prevented by a single dose of MitoQ bound to nanoparticles, which were shown to localise and prevent oxidative stress in the placenta but not in the fetus. We suggest the possibility of developing preventative treatments that target the placenta and not the fetus to reduce risk of psychiatric disease in later life.

Prenatal hypoxia and placental oxidative stress: linkages to developmental origins of cardiovascular disease.

Intrauterine growth restriction (IUGR, a pregnancy complication where the fetus does not reach its genetic growth potential) is a leading cause of fetal morbidity and mortality with a significant impact on population health. IUGR is associated with gestational hypoxia; which can lead to placental oxidative stress and fetal programming of cardiovascular disease. Mitochondria are a major source of placental oxidative stress and may provide a therapeutic target to mitigate the detrimental effects of placental oxidative stress on pregnancy outcomes. A nanoparticle-mediated delivery of a mitochondrial antioxidant to the placenta is a potential novel approach that may avoid unwanted off-target effects on the developing offspring.

MicroRNA-210 Targets Ten-Eleven Translocation Methylcytosine Dioxygenase 1 and Suppresses Pregnancy-Mediated Adaptation of Large Conductance Ca2+-Activated K+ Channel Expression and Function in Ovine Uterine Arteries.

Gestational hypoxia inhibits large conductance Ca2+-activated K+ (BKCa) channel expression and function in uterine arterial adaptation to pregnancy. Given the findings that microRNA-210 (miR-210) is increased in hypoxia during gestation and preeclampsia, the present study sought to investigate the role of miR-210 in the regulation of BKCa channel adaptation in the uterine artery. Gestational hypoxia significantly increased uterine vascular resistance and blood pressure in pregnant sheep and upregulated miR-210 in uterine arteries. MiR-210 bound to ovine ten-eleven translocation methylcytosine dioxygenase 1 mRNA 3' untranslated region and decreased ten-eleven translocation methylcytosine dioxygenase 1 mRNA and protein abundance in uterine arteries of pregnant sheep, as well as abrogated steroid hormone-induced upregulation of ten-eleven translocation methylcytosine dioxygenase 1 expression in uterine arteries of nonpregnant animals. In accordance, miR-210 blocked pregnancy- and steroid hormone-induced upregulation of BKCa channel β1 subunit expression in uterine arteries. Functionally, miR-210 suppressed BKCa channel current density in uterine arterial myocytes of pregnant sheep and inhibited steroid hormone-induced increases in BKCa channel currents in uterine arteries of nonpregnant animals. Blockade of endogenous miR-210 inhibited hypoxia-induced suppression of BKCa channel activity. In addition, miR-210 decreased BKCa channel-mediated relaxations and increased pressure-dependent myogenic tone of uterine arteries. Together, the results demonstrate that miR-210 plays an important role in the downregulation of ten-eleven translocation methylcytosine dioxygenase 1 and repression of BKCa channel function in uterine arteries, revealing a novel mechanism of epigenetic regulation in the maladaptation of uterine hemodynamics in gestational hypoxia and preeclampsia.

Maternal chronic hypoxia increases expression of genes regulating lung liquid movement and surfactant maturation in male fetuses in late gestation.

Chronic fetal hypoxaemia is a common pregnancy complication associated with intrauterine growth restriction that may influence respiratory outcome at birth. We investigated the effect of maternal chronic hypoxia for a month in late gestation on signalling pathways regulating fetal lung maturation and the transition to air-breathing at birth using isobaric hypoxic chambers without alterations to maternal food intake. Maternal chronic hypoxia in late gestation increases fetal lung expression of genes regulating hypoxia signalling, lung liquid reabsorption and surfactant maturation, which may be an adaptive response in preparation for the successful transition to air-breathing at birth. In contrast to other models of chronic fetal hypoxaemia, late gestation onset fetal hypoxaemia promotes molecular regulation of fetal lung maturation. This suggests a differential effect of timing and duration of fetal chronic hypoxaemia on fetal lung maturation, which supports the heterogeneity observed in respiratory outcomes in newborns following exposure to chronic hypoxaemia in utero. Chronic fetal hypoxaemia is a common pregnancy complication that may arise from maternal, placental and/or fetal factors. Respiratory outcome of the infant at birth likely depends on the duration, timing and severity of the hypoxaemic insult. We have isolated the effect of maternal chronic hypoxia (MCH) for a month in late gestation on fetal lung development. Pregnant ewes were exposed to normoxia (21% O2 ) or hypoxia (10% O2 ) from 105 to 138days of gestation (term ∼145days). At 138days, gene expression in fetal lung tissue was determined by quantitative RT-PCR. Cortisol concentrations were determined in fetal plasma and lung tissue. Numerical density of surfactant protein positive cells was determined by immunohistochemistry. MCH reduced maternal PaO2 (106±2.9vs. 47±2.8mmHg) and fetal body weight (4.0±0.4vs. 3.2±0.9kg). MCH increased fetal lung expression of the anti-oxidant marker CAT and decreased expression of the pro-oxidant marker NOX-4. MCH increased expression of genes regulating hypoxia signalling and feedback (HIF-3α, KDM3A, SLC2A1, EGLN-3). There was no effect of MCH on fetal plasma/lung tissue cortisol concentrations, nor genes regulating glucocorticoid signalling (HSD11B-1, HSD11B-2, NR3C1, NR3C2). MCH increased expression of genes regulating sodium (SCNN1-B, ATP1-A1, ATP1-B1) and water (AQP-4) movement in the fetal lung. MCH promoted surfactant maturation (SFTP-B, SFTP-D, ABCA3) at the molecular level, but did not alter the numerical density of surfactant positive cells in lung tissue. MCH in late gestation promotes molecular maturation of the fetal lung, which may be an adaptive response in preparation for the successful transition to air-breathing at birth.

Влияние острой гипоксии в позднем пренатальном периоде на проявление нейротоксического эффекта толуола у взрослых белых крыс

The aim of the present investigation is to estimate the role of prenatal hypoxia in toluene neurotoxic effect in adult rats. Toluene-induced behavioral and electroencephalographic manifestations were investigated in animals with normal and abnormal embryonic development. To simulate prenatal hypoxia, we gave subcutaneous injections of sodium nitrite to pregnant female rats in a dose of 50mg/kg on the 18–19th day of gestation. At the age of 3months the males from the offspring were exposed to toluene inhalation (560mg/m3, 4w eeks, 4h/day, 5days/week). After toluene inhalation exposure we estimated rats’ individual behavior by plus maze test and visual and auditory evoked potentials (VEPs and AEPs). Toluene reduced P2N2 interpeak amplitude of VEPs compared with control rats without any latency change. We found out that toluene exposure of rats with acute prenatal hypoxia in late gestation had led to inhibition of motor activity and a statistically significant increase in latency of VEP’s N1, AEP’s P3 and N1P2 interpeak amplitude of AEP in comparison with all remained groups. Thus, these results show that prenatal hypoxic damage to the central nervous system is an aggravating factor in toluene intoxication in rats.

Gestational stress induces the unfolded protein response, resulting in heart defects.

Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD.

The Application of Iodine‐Enhanced Micro‐CT Scanning Protocols to Analyze Multiple Tissue Types and Organ Systems in Mouse Embryos with Short‐term Gestational Hypoxia

We have previously demonstrated that mutations in Hes7 and Mesp2 genes, coupled with short‐term gestational hypoxia, significantly increase the range and severity of vertebral defects in a mouse model (Sparrow et al., 2012). In addition, disruptions of the Notch pathway result not only in vertebral, but also muscle defects (Fisher et al., 2012). These studies utilized skeletal preparations to document the range of vertebral defects, while micro‐dissections were conducted to investigate muscle defects. These techniques are relatively labor‐intensive and not amenable to analyses of multiple tissue types and organ systems within each specimen. In order to facilitate the analysis of multiple tissues in whole embryos, we investigated the utility of iodine‐enhanced microCT scanning protocols. A series of experiments were conducted to determine the optimal percentage concentration and duration of staining for 17.5‐day‐old mouse embryos. Embryos were fixed in 4% paraformaldehyde and subsequently stained with various concentrations of Lugol's iodine solution (25%, 50%, 75%, 100%), for different lengths of time (2, 5, 10 days). Images were acquired using a SKYSCAN 1172 micro‐CT scanner, with an X‐ray source at 70kVp and 142 μA, or 55kVp and 181 μA, using a 0.5 mm aluminum filter. The iodine staining protocols did result in visualization of a diversity of soft‐tissue structures including skeletal muscle, blood vessels, and internal organs. However, the specific parameters of the staining protocols greatly impacted the quality of the images, with low concentrations (25%) resulting in poor resolution of soft tissue and high concentrations (100%) resulting in tissue deformation. Results also indicate that the duration of staining as well as the delivery of the stain (one dose versus a step‐wise progression) may affect outcomes. Despite these challenges, this study suggests that iodine‐enhanced micro‐CT scanning protocols can be utilized to conduct whole embryo analyses of defects related to hypoxic conditions during development.Support or Funding InformationFunding for this project was provided by the American Association of Anatomists and the University of Arizona.

Antenatal hypoxia induces epigenetic repression of glucocorticoid receptor and promotes ischemic-sensitive phenotype in the developing heart

Large studies in humans and animals have demonstrated a clear association of an adverse intrauterine environment with an increased risk of cardiovascular disease later in life. Yet mechanisms remain largely elusive. The present study tested the hypothesis that gestational hypoxia leads to promoter hypermethylation and epigenetic repression of the glucocorticoid receptor (GR) gene in the developing heart, resulting in increased heart susceptibility to ischemia and reperfusion injury in offspring. Hypoxic treatment of pregnant rats from day 15 to 21 of gestation resulted in a significant decrease of GR exon 14, 15, 16, and 17 transcripts, leading to down-regulation of GR mRNA and protein in the fetal heart. Functional cAMP-response elements (CREs) at −4408 and −3896 and Sp1 binding sites at −3425 and −3034 were identified at GR untranslated exon 1 promoters. Hypoxia significantly increased CpG methylation at the CREs and Sp1 binding sites and decreased transcription factor binding to GR exon 1 promoter, accounting for the repression of the GR gene in the developing heart. Of importance, treatment of newborn pups with 5-aza-2′-deoxycytidine reversed hypoxia-induced promoter methylation, restored GR expression and prevented hypoxia-mediated increase in ischemia and reperfusion injury of the heart in offspring. The findings demonstrate a novel mechanism of epigenetic repression of the GR gene in fetal stress-mediated programming of ischemic-sensitive phenotype in the heart.

Gestational hypoxia modulates expression of corticotropin-releasing hormone and arginine vasopressin in the paraventricular nucleus in the ovine fetus.

Maturation of the fetal hypothalamo-pituitary-adrenocortical (HPA) axis is critical for organ maturation necessary for the fetus to transition to the ex-utero environment. Intrauterine stressors can hasten maturation of the HPA axis leading to fetal growth restriction and in sheep, premature birth. We have previously reported that high-altitude mediated, long-term-moderate gestational hypoxia (LTH) during gestation has a significant impact on the fetal HPA axis. Significant effects were observed at the level of both the anterior pituitary and adrenal cortex resulting in elevated plasma ACTH during late gestation with decreased adrenocortical expression of enzymes rate limiting for cortisol synthesis. As such, these fetuses exhibited the normal ontogenic rise in fetal plasma cortisol but an exaggerated cortisol response to acute stress. This study extended these findings to ACTH secretagogue expression in the PVN using insitu hybridization. We report that the expression of AVP but not CRH was increased in the medial parvocellular PVN (mpPVN) in the LTH fetus. This represented an increase in both AVP mRNA per neuron as well as an increase in AVP hybridizing neurons with no increase in mpPVN CRH neurons. LTH had no effect on PVN volume, area of CRH or AVP hybridization, thus LTH did not have a trophic effect on the size of the nucleus. In conclusion, there appears to be a switch from CRH to AVP as a primary ACTH secretagogue in response to LTH, supporting our previous findings of increased anterior pituitary sensitivity to AVP over CRH in the LTH fetus.

Direct effect of chronic hypoxia in suppressing large conductance Ca(2+)-activated K(+) channel activity in ovine uterine arteries via increasing oxidative stress.

Chronic hypoxia has a direct effect in down-regulating the BKCa channel β1 subunit and inhibiting the BKCa channel activity in uterine arteries of pregnant sheep. Oxidative stress plays a causal role in hypoxia-mediated suppression of BKCa channel function. The steroid hormone-induced effect on BKCa channels is a target of hypoxia-mediated oxidative stress. Inhibition of oxidative stress ameliorates the adverse effect of hypoxia both ex vivo and in vivo in pregnant sheep exposed to long-term high-altitude hypoxia. Our findings provide novel evidence of a causative role of oxidative stress in hypoxia-mediated inhibition of the BKCa channel activity in uterine arteries and new insights in understanding and alleviating pregnancy complications associated with gestational hypoxia such as pre-eclampsia and fetal growth restriction. Uterine arteries of pregnant sheep acclimatized to long-term high-altitude hypoxia were associated with a decrease in large-conductance Ca(2+)-activated K(+) (BKCa) channel activity. The present study tested the hypothesis that prolonged hypoxia has a direct effect in suppressing BKCa channel activity by increasing oxidative stress. Uterine arteries were isolated from non-pregnant and near-term (∼142 days) pregnant sheep, and were treated ex vivo with 21.0 or 10.5% O2 for 48 h. The hypoxia treatment significantly increased the production of reactive oxygen species in uterine arteries, which was blocked by N-acetylcysteine. In uterine arteries of pregnant sheep, hypoxia significantly inhibited BKCa channel current density, decreased NS1619-induced relaxations and increased pressure-dependent tone, which were annulled by N-acetylcysteine. In accordance, hypoxia resulted in down-regulation of BKCa channel β1 subunit, which was restored in the presence of N-acetylcysteine. In addition, the N-acetylcysteine treatment significantly increased BKCa channel β1 subunit abundance and BKCa channel current density in uterine arteries from pregnant sheep exposed to high-altitude hypoxia (3801 m, PaO2: 60 mmHg) for 110 days. In uterine arteries of non-pregnant animals, hypoxia inhibited steroid hormone-induced up-regulation of BKCa channel current density and NS1619-mediated relaxations, which were reversed by N-acetylcysteine. Furthermore, the synthetic superoxide dismutase and catalase mimetic EUK-134 also ablated the effects of hypoxia on BKCa channel currents in uterine arteries. The results demonstrate a direct effect of hypoxia in inhibiting the BKCa channel activity in uterine arteries via increased oxidative stress.

Late gestational hypoxia and a postnatal high salt diet programs endothelial dysfunction and arterial stiffness in adult mouse offspring.

Gestational hypoxia and high dietary salt intake have both been associated with impaired vascular function in adulthood. Using a mouse model of prenatal hypoxia, we examined whether a chronic high salt diet had an additive effect in promoting vascular dysfunction in offspring. Pregnant CD1 dams were placed in a hypoxic chamber (12% O2) or housed under normal conditions (21% O2) from embryonic day 14.5 until birth. Gestational hypoxia resulted in a reduced body weight for both male and female offspring at birth. This restriction in body weight persisted until weaning, after which the animals underwent catch-up growth. At 10 weeks of age, a subset of offspring was placed on a high salt diet (5% NaCl). Pressurized myography of mesenteric resistance arteries at 12 months of age showed that both male and female offspring exposed to maternal hypoxia had significantly impaired endothelial function, as demonstrated by impaired vasodilatation to ACh but not sodium nitroprusside. Endothelial dysfunction caused by prenatal hypoxia was not exacerbated by postnatal consumption of a high salt diet. Prenatal hypoxia increased microvascular stiffness in male offspring. The combination of prenatal hypoxia and a postnatal high salt diet caused a leftward shift in the stress-strain relationship in both sexes. Histopathological analysis of aortic sections revealed a loss of elastin integrity and increased collagen, consistent with increased vascular stiffness. These results demonstrate that prenatal hypoxia programs endothelial dysfunction in both sexes. A chronic high salt diet in postnatal life had an additive deleterious effect on vascular mechanics and structural characteristics in both sexes.

Long-Term Gestational Hypoxia Modulates Expression of Key Genes Governing Mitochondrial Function in the Perirenal Adipose of the Late Gestation Sheep Fetus.

We previously reported that long-term hypoxia (LTH) increases expression of brown adipose tissue (BAT) genes in the perirenal adipose in the ovine fetus. The mechanisms with which hypoxia mediates the enhanced BAT phenotype are unresolved. This study was designed to examine the effects of LTH on (1) the expression of endothelial cell nitric oxide synthase (eNOS) and (2) indicators of mitochondrial biogenesis (transcription factors mitochondrial transcription factor A (mtTFA), nuclear respiratory factor (NRF) 1, and NRF-2; cytochrome c oxidase (COX) I, II, and IV and mitochondrial DNA content). Pregnant ewes were maintained at high altitude (3820 m) from ∼40 to 137 to 140 days of gestation and perirenal adipose was collected from normoxic control and LTH fetuses. There was no effect of LTH on fetal body weight or perirenal adipose mass. Long-term hypoxia increased (P < .05) perirenal eNOS and phospho-eNOS, messenger RNA (mRNA) for NRF1, NRF-2, mtTFA as well as COX-I, COX-II, and COX-IV mRNA. In contrast, mRNA for 2 markers for cellular proliferation (Ki67 and proliferating cell nuclear antigen [PCNA]) was lower in perirenal adipose from LTH fetuses compared to controls (P < .05), while mitochondrial to nuclear DNA ratio did not differ between groups. In conclusion, nitric oxide may function as a mechanism via which LTH enhances the BAT phenotype in fetal sheep prior to birth. Although there is an apparent increase in genes supporting mitochondrial function and adaptive thermogenesis in response to LTH, there does not appear to be an increased mitochondrial biogenesis per se. Such adaptive changes may provide a mechanism for the prominence of the BAT phenotype observed in the late gestation LTH fetus.

Renal developmental defects resulting from in utero hypoxia are associated with suppression of ureteric β-catenin signaling

Gestational stressors, including glucocorticoids and protein restriction, can affect kidney development and hence final nephron number. Since hypoxia is a common insult during pregnancy, we studied the influence of oxygen tension on kidney development in models designed to represent a pathological hypoxic insult. In vivo mouse models of moderate, transient, midgestational (12% O₂, 48 h, 12.5 dpc) or severe, acute, early-gestational (5.5-7.5% O₂, 8 h, 9.5-10.5 dpc) hypoxia were developed. The embryo itself is known to mature under hypoxic conditions with embryonic tissue levels of oxygen estimated to be 5%-8% (physiological hypoxia) when the mother is exposed to ambient normoxia. Both in vivo models generated phenotypes seen in patients with congenital anomalies of the kidney and urinary tract (CAKUT). Severe, acute, early hypoxia resulted in duplex kidney, while moderate, transient, midgestational hypoxia permanently reduced ureteric branching and nephron formation. Both models displayed hypoxia-induced reductions in β-catenin signaling within the ureteric tree and suppression of the downstream target gene, Ccnd1. Thus, we show a link between gestational hypoxia and CAKUT, the phenotype of which varies with timing, duration, and severity of the hypoxic insult.

Impact of inhaled nitric oxide on white matter damage in growth-restricted neonatal rats.

Fetal growth restriction is the second leading cause of perinatal morbidity and mortality, and neonates with intrauterine growth retardation (IUGR) have increased neurocognitive and neuropsychiatric morbidity. These neurocognitive impairments are mainly related to injury of the developing brain associated with IUGR. Growing evidence from preclinical models of brain injury in both adult and neonatal rodents supports the view that nitric oxide can promote neuroprotection. In a model of IUGR induced by protracted gestational hypoxia leading to diffuse white matter injury, we subjected neonatal rats to low dose (5 ppm) but long-lasting (7 d) exposure to inhaled NO (iNO). We used a combination of techniques, including immunohistochemistry, quantitative PCR, and cognitive assessment, to assess neuroprotection. Antenatal hypoxia-induced IUGR was associated with severe neuroinflammation and delayed myelination. iNO exposure during the first postnatal week significantly attenuated cell death and microglial activation, enhanced oligodendroglial proliferation and finally improved myelination. Remarkably, iNO was associated with the specific upregulation of P27kip1, which initiates oligodendrocytic differentiation. Finally, iNO counteracted the deleterious effects of hypoxia on learning abilities. This study provides new evidence that iNO could be effective in preventing brain damage and/or enhancing repair of the developing brain.

Epigenetic upregulation of large-conductance Ca2+-activated K+ channel expression in uterine vascular adaptation to pregnancy.

Our previous study demonstrated that pregnancy increased large-conductance Ca(2+)-activated potassium channel β1 subunit (BKβ1) expression and large-conductance Ca(2+)-activated potassium channel activity in uterine arteries, which were abrogated by chronic hypoxia. The present study tested the hypothesis that promoter methylation/demethylation is a key mechanism in epigenetic reprogramming of BKβ1 expression patterns in uterine arteries. Ovine BKβ1 promoter of 2315 bp spanning from -2211 to +104 of the transcription start site was cloned, and an Sp1-380 binding site that contains CpG dinucleotide in its core binding sequences was identified. Site-directed deletion of the Sp1 site significantly decreased the BKβ1 promoter activity. Estrogen receptor-α bound to the Sp1 site through tethering to Sp1 and upregulated the expression of BKβ1. The Sp1 binding site at BKβ1 promoter was highly methylated in uterine arteries of nonpregnant sheep, and methylation inhibited transcription factor binding and BKβ1 promoter activity. Pregnancy caused a significant decrease in CpG methylation at the Sp1 binding site and increased Sp1 binding to the BKβ1 promoter and BKβ1 mRNA abundance. Chronic hypoxia during gestation abrogated this pregnancy-induced demethylation and upregulation of BKβ1 expression. The results provide evidence of a novel mechanism of promoter demethylation in pregnancy-induced reprogramming of large-conductance Ca(2+)-activated potassium channel expression and function in uterine arteries and suggest new insights of epigenetic mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.