Martha C. Tissot van Patot

Human placental metabolic adaptation to chronic hypoxia, high altitude: hypoxic preconditioning

We have previously demonstrated placentas from laboring deliveries at high altitude have lower binding of hypoxia-inducible transcription factor (HIF) to DNA than those from low altitude. It has recently been reported that labor causes oxidative stress in placentas, likely due to ischemic hypoxic insult. We hypothesized that placentas of high-altitude residents acquired resistance, in the course of their development, to oxidative stress during labor. Full-thickness placental tissue biopsies were collected from laboring vaginal and nonlaboring cesarean-section term (37–41 wk) deliveries from healthy pregnancies at sea level and at 3,100 m. After freezing in liquid nitrogen within 5 min of delivery, we quantified hydrophilic and lipid metabolites using 31P and 1H NMR metabolomics. Metabolic markers of oxidative stress, increased glycolysis, and free amino acids were present in placentas following labor at sea level, but not at 3,100 m. In contrast, at 3,100 m, the placentas were characterized by the presence of concentrations of stored energy potential (phosphocreatine), antioxidants, and low free amino acid concentrations. Placentas from pregnancies at sea level subjected to labor display evidence of oxidative stress. However, laboring placentas at 3,100 m have little or no oxidative stress at the time of delivery, suggesting greater resistance to ischemia-reperfusion. We postulate that hypoxic preconditioning might occur in placentas that develop at high altitude.

Hypoxia: adapting to high altitude by mutating EPAS-1, the gene encoding HIF-2α.

Living at high altitude is demanding and thus drives adaptational mechanisms. The Tibetan population has had a longer evolutionary period to adapt to high altitude than other mountain populations such as Andeans. As a result, some Tibetans living at high altitudes do not show markedly elevated red blood cell production as compared to South American high altitude natives such as Quechuas or Aymaras, thereby avoiding high blood viscosity creating cardiovascular risk. Unexpectedly, the responsible mutation(s) reducing red blood cell production do not involve either the gene encoding the blood hormone erythropoietin (Epo), or the corresponding regulatory sequences flanking the Epo gene. Similarly, functional mutations in the hypoxia-inducible transcription factor 1α (HIF-1α) gene that represents the oxygen-dependent subunit of the HIF-1 heterodimer, the latter being the main regulator of over 100 hypoxia-inducible genes, have not been described so far. It was not until very recently that three independent groups showed that the gene encoding HIF-2α, EPAS-1 (Wenger et al. 1997), represents a key gene mutated in Tibetan populations adapted to living at high altitudes (Beall et al. 2010 , Yi et al. 2010 , Simonson et al. 2010). Hypoxia-inducible transcription factors were first identified by the description of HIF-1 (Semenza et al. 1991 , 1992), which was subsequently found to enhance transcription of multiple genes that encode proteins necessary for rescuing from hypoxic exposure, including erythropoietic, angiogenic and glycolytic proteins. Then HIF-2 was identified (Ema et al. 1997 ; Flamme et al. 1997 ; Hogenesch et al. 1997 ; and Tian et al. 1997) and although it is highly similar to HIF-1 and has the potential to bind (Camenisch et al. 2001) and mediate (Mole et al. 2009) many of the same genes as HIF-1, its biological actions in response to hypoxia are distinct from those of HIF-1 (reviewed by Loboda et al. 2010). By now, several of these HIF-2 mediated processes have been implicated in the human response to high altitude exposure including erythropoiesis (Kapitsinou et al. 2010), iron homeostasis (Peyssonnaux et al. 2008), metabolism (Shohet et al. 2007; Tormos et al. 2010; Biswas et al. 2010 ; Rankin et al. 2009) and vascular permeability (Chen et al. 2009; Tanaka et al. 2005), among others. Clearly, mutation of EPAS-1 has the potential to bring far more advantage when adapting to high altitude than solely mutating the Epo gene.

Prophylactic low-dose acetazolamide reduces the incidence and severity of acute mountain sickness.

Previous studies have shown low-dose acetazolamide to be effective in preventing AMS in persons already at high altitude and then moving higher, a relatively low risk situation. We wished to evaluate prophylactic administration of low-dose acetazolamide for reducing the incidence and severity of AMS in a high-risk setting: rapid ascent from 1600 to 4300 m. We performed a double-blind, randomized, placebo-controlled study with human subjects (n=44) exposed to 4300 m for 24 h. Subjects were treated for 3 days prior to ascent to 4300 m and during day 1 at altitude with placebo (n=22) or acetazolamide 250 mg/day (125 mg bid, n=22). AMS diagnosis required both an AMS-C score from the Environmental Symptom Questionnaire-III>or=0.7 and a Lake Louise Symptom (LLS) questionnaire score>or=3 plus headache. Acetazolamide reduced the incidence of AMS compared to placebo-treated subjects (14% vs. 45%, respectively, p=0.02), and the number needed to treat was 3. The AMS-C and LLS scores were lower in acetazolamide-treated subjects, indicating less severe AMS. Low-dose acetazolamide administered prior to ascent and on day 1 at 4300 m effectively reduced the incidence and severity of AMS in a high-risk setting.

Competitive Substrates for P-Glycoprotein and Organic Anion Protein Transporters Differentially Reduce Blood Organ Transport of Fentanyl and Loperamide: Pharmacokinetics and Pharmacodynamics in Sprague Dawley Rats

BACKGROUND: Drug transport proteins may be instrumental in controlling the concentration of fentanyl at &mgr; receptors in the brain and may provide potential therapeutic targets for controlling an individual response to opioid administration. P-glycoprotein (P-gp) efflux transporter and organic anion transport protein inward transporters (OATP, human; Oatp, rat) have been implicated in fentanyl and verapamil (only P-gp) transport across the blood–brain barrier. We hypothesized that transport proteins P-gp and Oatp mediate opioid uptake in a drug and organ-specific manner, making them excellent potential targets for therapeutic intervention. METHODS: Opioid (fentanyl or loperamide) was administered by IV infusion to Sprague-Dawley rats alone or in combination with competitive substrates of P-gp (verapamil) or Oatp (pravastatin, naloxone). Plasma, lung, and brain were collected over 10 min and at 60 min after opioid infusion and opioid concentration determined using liquid chromatography/mass spectrometry (LC/LC-MS/MS). Continuous electroencephalogram was used to determine the in vivo response to fentanyl and loperamide in the presence and absence of verapamil. RESULTS: Loperamide brain:plasma (PB) and lung:plasma (PL) partitioning was increased two and fivefold, respectively in the presence of verapamil. Verapamil administration was lethal unless the loperamide dose was reduced by half (0.95–0.475 mg/kg). Fentanyl brain:plasma and lung:plasma were reduced four and sixfold, respectively, by pravastatin and naloxone, whereas verapamil had much less effect. Electroencephalogram results indicated that verapamil reduced the fentanyl-induced central nervous system (CNS) effect and increased the loperamide CNS effect. CONCLUSION: Protein transporters appear to be organ and drug-specific in vivo, affecting first-pass pulmonary uptake and CNS response to opioid administration. Further, data suggest that transport protein inhibition may prove useful for normalizing an individual response to opioids.

HYPoXia and PlaCEntal REModElling

In the first trimester of pregnancy fetal trophoblast cells invade the maternal uterine spiral arteries leading to loss of the vascular cells from the vessel wall and remodelling of the extracellular matrix. This is crucial to ensure that sufficient blood can reach the developing fetus. Impaired arterial remodelling is a feature of the major pregnancy pathologies pre-eclampsia and fetal growth restriction. Despite its importance, little is known about the regulation of this process. We have shown, using in vitro culture models and ex vivo explant models, that trophoblast cells play an active role in remodelling spiral arteries, and have implicated apoptotic events in this process. Further we have shown that trophoblast-derived factors such as Fas-ligand, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) are important regulators of this process. The oxygen tension within the uteroplacental environment will vary with gestational age and will depend on the extent of trophoblast invasion and artery remodelling. Fluctuations in oxygen tension may be an important determinant of cellular events both during invasion towards uterine vessels and during the remodelling process. The components of this process known to be regulated by oxygen are reviewed, including lessons that can be learned from pregnancies at high altitude. In addition, data on the effect of varying oxygen tension on trophoblast production of pro-apoptotic factors and susceptibility of vascular smooth muscle cells to induction of apoptosis are described.

The neuronal control of hypoxic ventilation: erythropoietin and sexual dimorphism.

Using mice, we demonstrated that when oxygen supply is lowered, erythropoietin (Epo), the main regulator of red blood cell production, modulates the ventilatory response by interacting with central (brainstem) and peripheral (carotid bodies) respiratory centers. We showed that enhanced Epo levels in the brainstem increased the hypoxic ventilatory response, and that intracerebroventricular injection of an Epo antagonist (soluble Epo receptor) abolished the ventilatory acclimatization to hypoxia. More recently, we have found that the impact of Epo on ventilation occurs in a sex‐dependent manner. Keeping in mind that women are less susceptible to several respiratory sicknesses and syndromes than men, we suggest that Epo plays a key role in sexually‐dimorphic hypoxic ventilation. Accordingly, we foresee that Epo has a potential therapeutic use as treatment for hypoxia‐associated ventilatory diseases.

Ginkgo biloba Does—and Does Not—Prevent Acute Mountain Sickness

Abstract Objective.—To determine the efficacy of 2 different sources of Ginkgo biloba extract (GBE) in reducing the incidence and severity of acute mountain sickness (AMS) following rapid ascent to high altitude. Methods.—Two randomized, double-blind, placebo-controlled cohort studies were conducted in which participants were treated with GBE (240 mg·d−1) or placebo prior to and including the day of ascent from 1600 m to 4300 m (ascent in 2 hours by car). Acute mountain sickness was diagnosed if the Environmental Symptom Questionnaire III acute mountain sickness–cerebral (AMS-C) score was ≥0.7 and the Lake Louise Symptom (LLS) score was ≥3 and the participant reported a headache. Symptom severity was also determined by these scores. Results.—Results were conflicting: Ginkgo biloba reduced the incidence and severity of AMS compared to placebo in the first but not the second study. In the first study, GBE reduced AMS incidence (7/21) vs placebo (13/19) (P = .027, number needed to treat = 3), and it also reduced severity (AMS-C = 0.77 ± 0.26 vs 1.59 ± 0.27, P = .029). In the second study, GBE did not reduce incidence or severity of AMS (GBE 4/15 vs placebo 10/22, P = .247; AMS-C = 0.48 ± 0.13 vs 0.58 ± 0.11, P = .272). The primary difference between the 2 studies was the source of GBE. Conclusions.—The source and composition of GBE products may determine the effectiveness of GBE for prophylaxis of AMS.

Enhanced leukocyte HIF-1α and HIF-1 DNA binding in humans after rapid ascent to 4300 m

Hypoxia plays a crucial role in the pathogenesis of a multitude of diseases and clinical conditions such as cancer, diabetes, cardiovascular disease, stroke, pulmonary disease, inflammation, organ transplant, and wound healing. Investigations into the role of hypoxia-inducible transcription factor (HIF) in disease development have been conducted with the basic premise that HIF is activated in vivo during hypoxia in humans, yet this basic physiologic premise has never verified. Thus, we hypothesized that HIF-1 DNA binding would be enhanced in vivo in humans in response to acute global hypoxia. Fourteen human subjects were exposed to normoxia (1600 m) and hypoxia (4300 m, approximately 12% O(2)) in a hypobaric hypoxic chamber (8 h). HIF-1 DNA binding and HIF-1alpha protein were evaluated in circulating leukocytes. Oxidative markers were evaluated by plasma metabolomics using nuclear magnetic resonance and by urinary 15-F(2t)-isoprostane concentrations. Leukocyte HIF-1 DNA binding was increased (p=0.007) and HIF-1alpha was greater during hypoxia compared to normoxia. Circulating total glutathione was reduced by 35% (p=0.001), and lactate and succinate were increased by 29 and 158%, respectively (p=0.007 and 0.001), as were urinary 15-F(2t)-isoprostanes (p=0.037). HIF-1 DNA binding and HIF-1alpha were elevated in vivo in leukocytes of healthy human subjects exposed to 12% oxygen, in association with plasma and urinary markers of hypoxic stress.

Ventilatory responses to acute and chronic hypoxia are altered in female but not male Paskin-deficient mice

Proteins harboring a Per-Arnt-Sim (PAS) domain are versatile and allow archaea, bacteria, and plants to sense oxygen partial pressure, as well as light intensity and redox potential. A PAS domain associated with a histidine kinase domain is found in FixL, the oxygen sensor molecule of Rhizobium species. PASKIN is the mammalian homolog of FixL, but its function is far from being understood. Using whole body plethysmography, we evaluated the ventilatory response to acute and chronic hypoxia of homozygous deficient male and female PASKIN mice (Paskin-/-). Although only slight ventilatory differences were found in males, female Paskin-/- mice increased ventilatory response to acute hypoxia. Unexpectedly, females had an impaired ability to reach ventilatory acclimatization in response to chronic hypoxia. Central control of ventilation occurs in the brain stem respiratory centers and is modulated by catecholamines via tyrosine hydroxylase (TH) activity. We observed that TH activity was altered in male and female Paskin-/- mice. Peripheral chemoreceptor effects on ventilation were evaluated by exposing animals to hyperoxia (Dejours test) and domperidone, a peripheral ventilatory stimulant drug directly affecting the carotid sinus nerve discharge. Male and female Paskin-/- had normal peripheral chemosensory (carotid bodies) responses. In summary, our observations suggest that PASKIN is involved in the central control of hypoxic ventilation, modulating ventilation in a gender-dependent manner.

Ginkgo biloba for Prevention of Acute Mountain Sickness: Does It Work?

Tissot van Patot, Martha, Linda E. Keyes, Guy Leadbetter III, and Peter H. Hackett. Ginkgo biloba for the prevention of acute mountain sickness: does it work? High Alt. Med. Biol. 10:00-00, 2009.-We review the current literature regarding the prophylactic use of Ginkgo biloba extract (GBE) in acute mountain sickness (AMS). We compare studies with regard to GBE dose, composition, study design, altitude reached, ascent rate, exercise, and risk of AMS. We then review what is known about the active components of GBE and their biological effects and apply this knowledge to interpret the results of AMS prevention trials. Overall, the literature suggests that due to the complexity of GBE the standardization of the product is inadequate, which likely explains the disparate clinical results. The variability in commercially available GBE products makes it impossible to determine whether GBE is truly effective for preventing or ameliorating AMS. However, investigating the roles of specific active components of GBE in the prevention of AMS could yield rewards both clinically and in our understanding of the pathophysiology of AMS.