Long-term High-altitude Hypoxia Research Articles

Effects of Long-Term Exposure to High Altitude Hypoxia on Cognitive Function and Its Mechanism: A Narrative Review.

Cognitive function is affected by low pressure and hypoxia in high-altitude environments, and is regulated by altitude and exposure time. With the economic development in the Qinghai-Tibet Plateau, the increase in work and study activities, as well as the development of plateau tourism, mountaineering, and other activities, the number of plateau immigrants is increasing daily. Long-term hypoxia challenges human physical and mental health, restricts work efficiency, and thus affects plateau economic development and human wellbeing. Therefore, it is of scientific and social significance to study how long-term exposure to the hypoxic plateau environment affects the physical and mental health of lowlanders as part of the ongoing development of the current plateau region. In this paper, we reviewed the research progress and mechanism of the effects of long-term (≥1 year) high-altitude (>2500 m) hypoxia exposure on the cognitive function of lowlanders, and suggested that the scope and sample size of the research should be expanded in the future, and that follow-up studies should be carried out to explore the time threshold of cognitive impairment and its compensatory or repair mechanism.

Urinary metabolic modulation in human participants residing in Siachen: a 1H NMR metabolomics approach

The main physiological challenge in high altitude environment is hypoxia which affects the aerobic metabolism reducing the energy supply. These changes may further progress toward extreme environment-related diseases. These are further reflected in changes in small molecular weight metabolites and metabolic pathways. In the present study, metabolic changes due to chronic environmental hypoxia were assessed using 1H NMR metabolomics by analysing the urinary metabolic profile of 70 people at sea level and 40 people at Siachen camp (3700 m) for 1 year. Multivariate statistical analysis was carried out, and PLSDA detected 15 metabolites based on VIP score > 1. ROC analysis detected cis-aconitate, Nicotinamide Mononucleotide, Tyrosine, Choline and Creatinine metabolites with a high range of sensitivity and specificity. Pathway analysis revealed 16 pathways impact > 0.05, and phenylalanine tyrosine and tryptophan biosynthesis was the most prominent altered pathway indicating metabolic remodelling to meet the energy requirements. TCA cycle, Glycine serine and Threonine metabolism, Glutathione metabolism and Cysteine alterations were other metabolic pathways affected during long-term high-altitude hypoxia exposure. Present findings will help unlock a new dimension for the potential application of NMR metabolomics to address extreme environment-related health problems, early detection and developing strategies to combat high altitude hypoxia.

Gestational High‐Altitude Hypoxia and Metabolomic Reprogramming in Pulmonary Arteries from Fetal Sheep

Gestational high‐altitude hypoxia increases the risk of myriad diseases in human infants including pulmonary hypertension of the newborn. Fetal sheep lungs are susceptible to long‐term intrauterine hypoxia, with the lung structure and function exhibiting characteristics associated with the development of pulmonary hypertension. Previous studies showed thickening of the pulmonary arterial medial wall, dysregulation of arterial reactivity, and reduced right ventricular output in fetal lambs following exposure to high altitude long‐term hypoxia. To further explore the mechanisms associated with hypoxia‐induced aberrations in the fetal sheep lung, we examined the premise that metabolomic and proteomic changes are associated with phenotypic transformation caused by intrauterine hypoxia. To address this hypothesis, we performed metabolomic and proteomic analysis on pulmonary arteries isolated from near term fetal lambs that were exposed to intrauterine high‐altitude hypoxia (3801 m) for the latter 110+ days of pregnancy or those gestated near sea level. The data demonstrated gestational hypoxia caused a glycolytic shift, reduced fatty acid oxidation, and elevated pentose phosphate metabolism along with increased histone expression, illustrative of increases in nucleotide synthesis. Increased nucleotide synthesis in response to gestational hypoxia is suggestive of a shift towards a proliferative and synthetic phenotype, providing insight into the mechanisms related to gestational hypoxia‐mediated pulmonary remodeling and hypertension. These studies begin to shed light on the mechanistic underpinnings associated with the phenotypic transformations to the pulmonary vasculature that occur with intrauterine hypoxia.Support or Funding InformationSupported in part by NIH grants HD083132 (LZ), 1U24DK097154 (OF ) through a pilot project grant to SMW and HD098477 (SMW). Additional support was provided by USDA Intramural projects #2032‐51530‐022‐00D and #2032‐51530‐025‐00D (J.W.N.). The USDA is an equal opportunity provider, and employer.

Long-Term High-Altitude Hypoxia and Alpha Adrenoceptor-Dependent Pulmonary Arterial Contractions in Fetal and Adult Sheep.

Autonomic innervation of the pulmonary vasculature triggers vasomotor contractility predominately through activation of alpha-adrenergic receptors (α-ARs) in the fetal circulation. Long-term hypoxia (LTH) modulates pulmonary vasoconstriction potentially through upregulation of α1-AR in the vasculature. Our study aimed to elucidate the role of α-AR in phenylephrine (PE)-induced pulmonary vascular contractility, comparing the effects of LTH in the fetal and adult periods on α-AR subtypes and PE-mediated Ca2+ responses and contractions. To address this, we performed wire myography, Ca2+ imaging, and mRNA analysis of pulmonary arteries from ewes and fetuses exposed to LTH or normoxia. Postnatal maturation depressed PE-mediated contractile responses. α2-AR activation contracted fetal vessels; however, this was suppressed by LTH. α1A- and α1B-AR subtypes contributed to arterial contractions in all groups. The α1D-AR was also important to contractility in fetal normoxic vessels and LTH mitigated its function. Postnatal maturity increased the number of myocytes with PE-triggered Ca2+ responses while LTH decreased the percentage of fetal myocytes reacting to PE. The difference between myocyte Ca2+ responsiveness and vessel contractility suggests that fetal arteries are sensitized to changes in Ca2+. The results illustrate that α-adrenergic signaling and vascular function change during development and that LTH modifies adrenergic signaling. These changes may represent components in the etiology of pulmonary vascular disease and foretell the therapeutic potential of adrenergic receptor antagonists in the treatment of pulmonary hypertension.

Long Term Hypoxia Reduces Levels of Oxylipins in Pulmonary Arteries and Venous Plasma of Fetal Sheep

High‐altitude long term hypoxia (LTH) during gestation compromises the development and function of the pulmonary vasculature in the fetus and later in the newborn. This may manifest as fetal growth restriction as well as pulmonary hypertension following birth. While the physiological regulation regarding the effects of hypoxia are well‐known, the dysregulation that occurs at the cellular and molecular level is not yet completely understood. Exploring the effects of LTH using a metabolomic approach is advantageous in deciphering the etiology associated with the development of disease. LTH is well regarded for inducing oxidative stress and causing inflammation. A number of possible regulators associated with LTH stress include oxylipins and endocannabinoids, products of polyunsaturated fatty acid (PUFA) oxidation. We hypothesized that high‐altitude LTH would reduce the levels of oxylipins and endocannabinoids of pulmonary arteries and plasma in fetal sheep. To test the hypothesis, we obtained samples of plasma and pulmonary arteries from fetal normoxic and hypoxic sheep raised at 3,800 m. altitude starting on gestation day 30. Metabolite levels were quantified using ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). Chemical similarity enrichment analyses and visualization by complex pathway analyses was performed on the datasets. Our results support our hypothesis that select oxylipin concentrations were diminished in both venous plasma and pulmonary arteries. Omega‐3 PUFAs alpha linolenic acid (ALA) and eicosapentaenoic acid (EPA) were reduced in both plasma and pulmonary arteries. 15‐HEPE and 12‐HEPE, derivatives of EPA and important anti‐inflammatory mediators, were also significantly reduced by LTH in plasma and pulmonary arterial samples from fetal animals. We believe that our studies provide insight toward identifying key biomarkers of LTH stress that will afford effective and prompt diagnosis of the neonate's physiological response. This is important when it comes to treating diseases associated with gestational hypoxia, which affects numerous mothers and newborns worldwide.Support or Funding InformationThis material is based upon work supported by NIH grants P01HD083132 (LZ) and 1U24DK097154 through a pilot project grant to SMW. VL is a recipient of the American Physiological Society's Short‐Term Research Education Program to Increase Diversity in Health‐Related Research (STRIDE) Fellowship funded by the APS and a grant from the National Heart, Lung and Blood Institute R25HL115473.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Acute Hypoxia Alters Ryanodine Receptor Activity in Pulmonary Arterial Myocytes of High Altitude Acclimatized Fetal and Adult Sheep

Intrauterine stress, such as long term high altitude hypoxia (LTH), can alter pulmonary function and reprogram fetal pulmonary vascular development, potentially causing respiratory distress and pulmonary hypertension after birth. Our recent data demonstrates that newborn LTH lambs exhibit exaggerated hypoxia‐induced pulmonary vasoconstriction (HPV). Previous evidence shows Ryanodine Receptor (RyR) activation is central to the HPV response. Furthermore, LTH disrupts RyR activated Ca2+ sparks in pulmonary arterial (PA) myocytes of sheep. Given the relationship between RyR and HPV responses, we tested the hypothesis that acute hypoxia (exposure to 4% O2) and LTH increase RyR mediated Ca2+ spark activity. This was tested by measuring the Ca2+ spark activity in PA myocytes of near‐term fetal lambs and adult sheep that resided at either low (335m, LA) or high (3801m, HA) altitude. Intracellular Ca2+ was recorded in myocytes of isolated PA tissues loaded with Fluo‐4 using line‐scan techniques on a confocal microscope. Line‐scan images were analyzed using custom software (SparkLab) to determine the number of cells displaying sparks, spark frequency, and spatial‐ temporal characteristics. Adult myocytes had greater prevalence and frequency of Ca2+ sparks relative to those of fetuses, confirming previous studies. Acute hypoxia significantly decreased spark prevalence and frequency in myocytes from both HA fetal and LA adult groups; however, acute hypoxia increased spark prevalence and frequency in HA adult sheep. Spark amplitude was decreased by acute hypoxia in the LA fetus but increased in the HA adult. Acute hypoxia also reduced spark duration in the fetus independent of altitude but increased the duration in HA adult. These results provide new insight into the influence of high altitude reprogramming of RyR function in PA of the fetus and adult and the roles their activity have during acute hypoxic exposures.Support or Funding InformationThis work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. JT was a Loma Linda University Summer Undergraduate Research Fellow while TY was a Walter E Macpherson Society Medical Student Summer Research Fellow.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long-term hypoxia uncouples Ca2+and eNOS in bradykinin-mediated pulmonary arterial relaxation

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca2+ with confocal microscopy. eNOS was inhibited with 100 μM NG-nitro-l-arginine methyl ester (l-NAME), PGI2 production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the β1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

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.

Antenatal Maternal Long-Term Hypoxia: Acclimatization Responses with Altered Gene Expression in Ovine Fetal Carotid Arteries

In humans and other species, long-term hypoxia (LTH) during pregnancy can lead to intrauterine growth restriction with reduced body/brain weight, dysregulation of cerebral blood flow (CBF), and other problems. To identify the signal transduction pathways and critical molecules, which may be involved in acclimatization to high altitude LTH, we conducted microarray with advanced bioinformatic analysis on carotid arteries (CA) from the normoxic near-term ovine fetus at sea-level and those acclimatized to high altitude for 110+ days during gestation. In response to LTH acclimatization, in fetal CA we identified mRNA from 38 genes upregulated >2 fold (P<0.05) and 9 genes downregulated >2-fold (P<0.05). The major genes with upregulated mRNA were SLC1A3, Insulin-like growth factor (IGF) binding protein 3, IGF type 2 receptor, transforming growth factor (TGF) Beta-3, and genes involved in the AKT and BCL2 signal transduction networks. Most genes with upregulated mRNA have a common motif for Pbx/Knotted homeobox in the promoter region, and Sox family binding sites in the 3′ un translated region (UTR). Genes with downregulated mRNA included those involved in the P53 pathway and 5-lipoxygenase activating proteins. The promoter region of all genes with downregulated mRNA, had a common 49 bp region with a binding site for DOT6 and TOD6, components of the RPD3 histone deacetylase complex RPD3C(L). We also identified miRNA complementary to a number of the altered genes. Thus, the present study identified molecules in the ovine fetus, which may play a role in the acclimatization response to high-altitude associated LTH.

Pulmonary artery smooth muscle cell proliferation and migration in fetal lambs acclimatized to high-altitude long-term hypoxia: role of histone acetylation

High-altitude long-term hypoxia (LTH) is known to induce pulmonary arterial smooth muscle cell (PASMC) proliferation in the fetus, leading to pulmonary arterial remodeling and pulmonary hypertension of the newborn. The mechanisms underlying these conditions remain enigmatic however. We hypothesized that epigenetic alterations in fetal PASMC induced by high-altitude LTH may play an important role in modulating their proliferation during pulmonary arterial remodeling. To test this hypothesis, we have analyzed epigenetic alterations in the pulmonary vasculature of fetal lambs exposed to high-altitude LTH [pregnant ewes were kept at 3,801 m altitude from ~40 to 145 days gestation] or to sea level atmosphere. Intrapulmonary arteries were isolated, and fetal PASMC were cultured from both control and LTH fetuses. Compared with controls, in LTH fetus pulmonary arteries measurements of histone acetylation and global DNA methylation demonstrated reduced levels of global histone 4 acetylation and DNA methylation, accompanied by the loss of the cyclin-dependent kinase inhibitor p21. Treatment of LTH fetal PASMCs with histone deacetylase (HDAC) inhibitor trichostatin A decreased their proliferation rate, in part because of altered expression of p21 at both RNA and protein level. In PASMC of LTH fetuses, HDAC inhibition also decreased PDGF-induced cell migration and ERK1/2 activation and modulated global DNA methylation. On the basis of these observations, we propose that epigenetic alterations (reduced histone acetylation and DNA methylation) caused by chronic hypoxia leads to fetal PASMC proliferation and vessel remodeling associated with vascular proliferative disease and that this process is regulated by p21.

Chronic Hypoxia During Gestation Causes Epigenetic Repression of the Estrogen Receptor-α Gene in Ovine Uterine Arteries via Heightened Promoter Methylation

Estrogen receptor-α (ERα) plays a key role in the adaptation of increased uterine blood flow in pregnancy. Chronic hypoxia is a common stress to maternal cardiovascular homeostasis and causes increased risk of preeclampsia. Studies in pregnant sheep demonstrated that hypoxia during gestation downregulated ERα gene expression in uterine arteries. The present study tested the hypothesis that hypoxia causes epigenetic repression of the ERα gene in uterine arteries via heightened promoter methylation. Ovine ERα promoter of 2035 bp spanning from −2000 to +35 of the transcription start site was cloned. No estrogen or hypoxia-inducible factor response elements were found at the promoter. Two transcription factor binding sites, USF −15 and Sp1 −520 , containing CpG dinucleotides were identified, which had significant effects on the promoter activity. The USF element binds transcription factors USF1 and USF2, and the Sp1 element binds Sp1, as well as ERα through Sp1. Deletion of the Sp1 site abrogated 17β-estradiol–induced increase in the promoter activity. In normoxic control sheep, CpG methylation at the Sp1 but not the USF site was significantly decreased in uterine arteries of pregnant as compared with nonpregnant animals. In pregnant sheep exposed to long-term high-altitude hypoxia, CpG methylation at both Sp1 and USF sites in uterine arteries was significantly increased. Methylation inhibited transcription factor binding and the promoter activity. The results provide evidence of hypoxia causing heightened promoter methylation and resultant ERα gene repression in uterine arteries and suggest new insights of molecular mechanisms linking gestational hypoxia to aberrant uteroplacental circulation and increased risk of preeclampsia.

Preservation of Serotonin-Mediated Contractility in Adult Sheep Pulmonary Arteries Following Long-Term High-Altitude Hypoxia

Long-term hypoxia (LTH) can increase serotonin (5-HT) signaling as well as extracellular calcium entry in adult rodent pulmonary arteries (PA), and 5-HT is associated with pulmonary hypertension. Because LTH, 5-HT, and calcium entry are related, we tested the hypothesis that LTH increases 5-HT-mediated PA contractility and associated calcium influx through L-type Ca2+ channels, nonselective cation channels (NSCC), and reverse-mode sodium-Ca2+ exchange. We performed wire myography and confocal calcium imaging on pulmonary arteries from adult ewes that lived near sea level or were maintained at high-altitude (3801 m) for ∼110 days. LTH did not increase the arterial medial wall thickness, nor did it affect the potency or efficacy for 5-HT-induced PA contraction. Ketanserin (100 nM), a 5-HT2A antagonist, shifted the 5-HT potency to a far greater extent than 1 μM GR-55562, a 5-HT1B/D inhibitor. These influences were unaffected by LTH. The rank order for reducing 5-HT-induced PA contraction in normoxic animals was extracellular calcium removal≈10 mM Ni2+≈10 μM verapamil≈10 μM nifedipine with 50 μM SKF 96365>30 μM KB-R7943≈100 μM flufenamic acid≈10 μM nifedipine≈100 μM Gd3+> 100 μM La3+>500 μM Ni2+≈10 μM diltiazem≈50 μM 2-APB≈100 μM LOE 908. Contraction was not reduced by 100 μM spermine or 30 μM SN-6. LTH increased the effects of KB-R7943 and mitigated those of nifedipine but did not affect calcium responses in imaging studies. Overall, in adult sheep, arterial structure and 5-HT2A and 5HT1B/D functions are preserved following LTH while the role of NSCC-related calcium-dependent contraction is increased. These elements indicate preservation of PA contractility in LTH with minimal functional changes.

Long-term maternal hypoxia: the role of extracellular Ca2+ entry during serotonin-mediated contractility in fetal ovine pulmonary arteries.

Antenatal maternal long-term hypoxia (LTH) can alter serotonin (5-HT) and calcium (Ca(2+)) signaling in fetal pulmonary arteries (PAs) and is associated with persistent pulmonary hypertension of the newborn (PPHN). In humans, the antenatal maternal hypoxia can be secondary to smoking, anemia, and chronic obstructive pulmonary disorders. However, the mechanisms of antenatal maternal hypoxia-related PPHN are unresolved. Because both LTH and 5-HT are associated with PPHN, we tested the hypothesis that antenatal maternal LTH can increase 5-HT-mediated PA contraction and associated extracellular Ca(2+) influx through L-type Ca(2+) channels (Ca(L)), nonselective cation channels (NSCCs), and reverse-mode sodium-calcium exchanger (NCX) in the near-term fetus. We performed wire myography and confocal-Ca(2+) imaging approaches on fetal lamb PA (∼ 140 days of gestation) from normoxic ewes or those acclimatized to high-altitude LTH (3801 m) for ∼110 days. Long-term hypoxia reduced the potency but not the efficacy of 5-HT-induced PA contraction. Ketanserin (100 nmol/L), a 5-HT(2A) antagonist, shifted 5-HT potency irrespective of LTH, while GR-55562 (1 µmol/L), a 5-HT(1B/D) inhibitor, antagonized 5-HT-induced contraction in normoxic fetuses only. Various inhibitors for Ca(L), NSCC, and reverse-mode NCX were used in contraction studies. Contraction was reliant on extracellular Ca(2+) regardless of maternal hypoxia, NSCC was more important to contraction than Ca(L), and reverse-mode NCX had little or no role in contraction. Long-term hypoxia also attenuated the effects of 2-APB and flufenamic acid and reduced Ca(2+) responses observed by imaging studies. Overall, LTH reduced 5HT(1B/D) function and increased NSCC-related Ca(2+)-dependent contraction in ovine fetuses, which may compromise pulmonary vascular function in the newborn.

Maturation and long-term hypoxia-induced acclimatization responses in PKC-mediated signaling pathways in ovine cerebral arterial contractility

In the developing fetus, cerebral arteries (CA) show striking differences in signal transduction mechanisms compared with the adult, and these differences are magnified in response to high-altitude long-term hypoxia (LTH). In addition, in the mature organism, cerebrovascular acclimatization to LTH may be associated with several clinical problems, the mechanisms of which are unknown. Because PKC plays a key role in regulating CA contractility, in fetal and adult cerebral arteries, we tested the hypothesis that LTH differentially regulates the PKC-mediated Ca(2+) sensitization pathways and contractility. In four groups of sheep [fetal normoxic (FN), fetal hypoxic (FH), adult normoxic (AN), and adult hypoxic (AH)], we examined, simultaneously, responses of CA tension and intracellular Ca(2+) concentration and measured CA levels of PKC, ERK1/2, RhoA, 20-kDa myosin light chain, and the 17-kDa PKC-potentiated myosin phosphatase inhibitor CPI-17. The PKC activator phorbol 12,13-dibutyrate (PDBu) produced robust contractions in all four groups. However, PDBu-induced contractions were significantly greater in AH CA than in the other groups. In all CA groups except AH, in the presence of MEK inhibitor (U-0126), the PDBu-induced contractions were increased a further 20-30%. Furthermore, in adult CA, PDBu led to increased phosphorylation of ERK1, but not ERK2; in fetal CA, the reverse was the case. PDBu-stimulated ERK2 phosphorylation also was significantly greater in FH than FN CA. Also, although RhoA/Rho kinase played a significant role in PDBu-mediated contractions of FN CA, this was not the case in FH or either adult group. Also, whereas CPI-17 had a significant role in adult CA contractility, this was not the case for the fetus. Overall, in ovine CA, the present study demonstrates several important maturational and LTH acclimatization changes in PKC-induced contractile responses and downstream pathways. The latter may play a key role in the pathophysiologic disorders associated with acclimatization to high altitude.

Role of Epidermal Growth Factor Receptor in Ovine Fetal Pulmonary Vascular Remodeling Following Exposure to High Altitude Long-Term Hypoxia

High altitude long-term hypoxia (LTH) in the fetus may result in pulmonary vascular smooth muscle cell (PVSMC) proliferation and pulmonary vascular remodeling. Our objective was to determine if epidermal growth factor receptor (EGFR) is involved in hypoxia induced PVSMC proliferation or in pulmonary vascular remodeling in ovine fetuses exposed to high altitude LTH. Fetuses of pregnant ewes that were held at 3820-m altitude from *30 to 140 days (LTH) gestation and sea level control pregnant ewes were delivered near term. Morphometric analyses and immunohistochemistry were done on fetal lung sections. Pulmonary arteries of LTH fetuses exhibited medial wall thickening and distal muscularization. Western blot analyses done on protein isolated from pulmonary arteries demonstrated an upregulation of EGFR. This upregulation was attributed in part to PVSMC in the medial wall by immunohistochemistry.Proliferation of fetal ovine PVSMC after 24 h of hypoxia (2% O2) was attenuated by inhibition of EGFR with 250 nmol tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), a specific EGFR protein tyrosine kinase inhibitor, when measured by [3H]-thymidine incorporation. Our data indicate that EGFR plays a role in fetal ovine pulmonary vascular remodeling following long-term fetal hypoxia and that inhibition of EGFR signaling may ameliorate hypoxia-induced pulmonary vascular remodeling.

Changes of Renal AT1/AT2 Receptors and Structures in Ovine Fetuses following Exposure to Long-Term Hypoxia

Background/Aims: The present study tested the hypothesis that chronic hypoxia adversely affects renal development in the ovine fetus. Methods: Kidneys were collected from near-term fetuses of pregnant ewes maintained at sea level or high altitude (3,801 m, PaO₂: approx. 60 mm Hg) for 110 days (n = 6 for each group). Results: Long-term high altitude hypoxia reduced the fetal kidney/body weight ratio. Histological analysis showed a significant enlargement in the Bowman’s space and swelling of tubule epithelial cells in the kidney of the hypoxic fetus. The histological alterations were limited to the cortical, but not medullary, zone. These alterations were associated with an increase in serum creatinine and a decrease in the BUN-to-creatinine ratio in hypoxic fetuses. Angiotensin II receptors (AT₁R and AT₂R) were detected in the glomerular and tubular regions of the kidney. Chronic hypoxia caused a significant increase in AT₁R and a decrease in AT₂R protein and mRNA abundance, resulting in a large increase in the AT₁R/AT₂R ratio in the fetal kidney. Conclusion: The results demonstrate an adverse effect of chronic hypoxia on renal AT₁R and AT₂R expression and functions in the fetus, suggesting a possible role of fetal hypoxia in the programming of renal diseases in fetal origins.

Effect of long-term high-altitude hypoxia on fetal pulmonary vascular contractility

Hypoxia in the fetus and/or newborn is associated with an increased risk of pulmonary hypertension. The present study tested the hypothesis that long-term high-altitude hypoxemia differentially regulates contractility of fetal pulmonary arteries (PA) and veins (PV) mediated by differences in endothelial NO synthase (eNOS). PA and PV were isolated from near-term fetuses of pregnant ewes maintained at sea level (300 m) or high altitude of 3,801 m for 110 days (arterial Po(2) of 60 Torr). Hypoxia had no effect on the medial wall thickness of pulmonary vessels and did not alter KCl-induced contractions. In PA, hypoxia significantly increased norepinephrine (NE)-induced contractions, which were not affected by eNOS inhibitor N(G)-nitro-l-arginine (l-NNA). In PV, hypoxia had no effect on NE-induced contractions in the absence of l-NNA. l-NNA significantly increased NE-induced contractions in both control and hypoxic PV. In the presence of l-NNA, NE-induced contractions of PV were significantly decreased in hypoxic lambs compared with normoxic animals. Acetylcholine caused relaxations of PV but not PA, and hypoxia significantly decreased both pD(2) and the maximal response of acetylcholine-induced relaxation in PV. Additionally, hypoxia significantly decreased the maximal response of sodium nitroprusside-induced relaxations of both PA and PV. eNOS was detected in the endothelium of both PA and PV, and eNOS protein levels were significantly higher in PV than in PA in normoxic lambs. Hypoxia had no significant effect on eNOS levels in either PA or PV. The results demonstrate heterogeneity of fetal pulmonary arteries and veins in response to long-term high-altitude hypoxia and suggest a likely common mechanism downstream of NO in fetal pulmonary vessel response to chronic hypoxia in utero.

Cerebral blood flow and oxygenation in ovine fetus: responses to superimposed hypoxia at both low and high altitude.

For the fetus, although the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (CBF) and cerebral oxygenation, the relation of CBF, cortical tissue P(O2) (tP(O2)), sagittal sinus P(O2), and related indices of cerebral oxygenation to arterial blood gases are not well defined. This is particularly true for that fetus subjected to long-term hypoxia (LTH). In an effort to elucidate these interrelations, we tested the hypothesis that in the fetus acclimatized to high altitude, cerebral oxygenation is not compromised relative to that at low altitude. By use of a laser Doppler flowmeter with a fluorescent O2 probe, in near-term fetal sheep at low altitude (n = 8) and those acclimatized to high altitude hypoxia (3801 m for 90 +/- 5 days; n = 6), we measured laser Doppler CBF (LD-CBF), tP(O2), and related variables in response to 40 min superimposed hypoxia. At both altitudes, fetal LD-CBF, cerebral O2 delivery, tP(O2), and several other variables including sagittal sinus P(O2), correlated highly with arterial P(O2) (P(a,O2)). In response to superimposed hypoxia (P(a,O2) = 11 +/- 1 Torr), LD-CBF was significantly blunted at high altitude, as compared with that at low altitude. In the two altitude groups fetal cerebral oxygenation was similar under both control conditions and with superimposed hypoxia, cortical tP(O2) decreasing from 8 +/- 1 and 6 +/- 1 Torr, respectively, to 2 +/- 1 Torr. Also, for these conditions sagittal sinus P(O2) and [HbO2] values were similar. In response to superimposed hypoxia, cerebral metabolic rate for O(2) decreased approximately 50% in each group (P < 0.05). For both the fetus at low altitude and that acclimatized to high altitude LTH, we present the first dose-response data on the relation of LD-CBF, cortical tP(O2), and sagittal sinus blood gas values to P(a,O2). In addition, despite differences in several variables, the fetus at high altitude showed evidence of successful acclimatization, supporting the hypothesis that such fetuses demonstrate no compromise in cerebral oxygenation.

Fetal cerebrovascular acclimatization responses to high-altitude, long-term hypoxia: a model for prenatal programming of adult disease?

During the past several decades, many risk factors for cerebrovascular and cardiovascular disease have been identified. More recently, it has been appreciated that inadequate nutrition and/or other intrauterine factors during fetal development may play an important role in the genesis of these conditions. An additional stress factor that may "program" the fetus for disease later in life is chronic hypoxia. In studies originally designed to examine the function of developing cerebral arterial function in response to long-term hypoxia (LTH), it has become clear that many cellular and subcellular changes may have important implications for later life. Here we review some of the significant alterations in fetal cerebral artery structure and function induced by high-altitude (3,820 m, 12,470 ft) LTH ( approximately 110 days). LTH is associated with augmentation or upregulation of presynaptic functions, including responses to perivascular (i.e., sympathetic) nerve stimulation, and structural maturational changes. In contrast, many postsynaptic functions related to the Ca(2+)-dependent contractile pathway tend to be downregulated, whereas elements of the Ca(2+)-independent contraction pathway are upregulated. The results emphasize the role of high-altitude LTH in modulating many aspects of electromechanical and pharmacomechanical coupling in the developing cerebral vasculature. A complicating factor is that the regulation of cerebrovascular tone by Ca(2+)-dependent and Ca(2+)-independent pathways changes significantly as a function of maturational age. In addition to highlighting independent regulation of various elements of the signal transduction cascade, the studies demonstrate the potential for LTH to program the fetus for cerebrovascular and other disease as an adult.