Limited role of the c-Jun N-terminal kinase pathway in a neonatal rat model of cerebral hypoxia-ischemia

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia-ischemia. Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase-3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic and autophagic. However the size of the lesion was unchanged. Further analysis showed that neonatal hypoxia-ischemia induced an immediate decrease in JNK phosphorylation (reflecting mainly P-JNK1) followed by a slow progressive increase (including P-JNK3 54kDa), whereas c-jun and c-fos expression were both strongly activated immediately after hypoxia-ischemia. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral hypoxia-ischemia in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Clear Cell Papillary Renal Cell Carcinoma and Renal Angiomyoadenomatous Tumor: Two Variants of a Morphologic, Immunohistochemical, and Genetic Distinct Entity of Renal Cell Carcinoma.

Clear cell papillary renal cell carcinoma (ccpRCC) and renal angiomyoadenomatous tumor (RAT) share morphologic similarities with clear cell (ccRCC) and papillary RCC (pRCC). It is a matter of controversy whether their morphologic, immunophenotypic, and molecular features allow the definition of a separate renal carcinoma entity. The aim of our project was to investigate specific renal immunohistochemical biomarkers involved in the hypoxia-inducible factor pathway and mutations in the VHL gene to clarify the relationship between ccpRCC and RAT. We investigated 28 ccpRCC and 9 RAT samples by immunohistochemistry using 25 markers. VHL gene mutations and allele losses were investigated by Sanger sequencing and fluorescence in situ hybridization. Clinical follow-up data were obtained for a subset of the patients. No tumor recurrence or tumor-related death was observed in any of the patients. Immunohistochemistry and molecular analyses led to the reclassification of 3 tumors as ccRCC and TFE3 translocation carcinomas. The immunohistochemical profile of ccpRCC and RAT samples was very similar but not identical, differing from both ccRCC and pRCC. Especially, the parafibromin and hKIM-1 expression exhibited differences in ccpRCC/RAT compared with ccRCC and pRCC. Genetic analysis revealed VHL mutations in 2/27 (7%) and 1/7 (14%) ccpRCC and RAT samples, respectively. Fluorescence in situ hybridization analysis disclosed a 3p loss in 2/20 (10%) ccpRCC samples. ccpRCC and RAT have a specific morphologic and immunohistochemical profile, but they share similarities with the more aggressive renal tumors. On the basis of our results, we regard ccpRCC/RAT as a distinct entity of RCCs.

Targeting cancer cell metabolism in pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer death by 2030. Current therapeutic options are limited, warranting an urgent need to explore innovative treatment strategies. Due to specific microenvironment constraints including an extensive desmoplastic stroma reaction, PDAC faces major metabolic challenges, principally hypoxia and nutrient deprivation. Their connection with oncogenic alterations such as KRAS mutations has brought metabolic reprogramming to the forefront of PDAC therapeutic research. The Warburg effect, glutamine addiction, and autophagy stand as the most important adaptive metabolic mechanisms of cancer cells themselves, however metabolic reprogramming is also an important feature of the tumor microenvironment, having a major impact on epigenetic reprogramming and tumor cell interactions with its complex stroma. We present a comprehensive overview of the main metabolic adaptations contributing to PDAC development and progression. A review of current and future therapies targeting this range of metabolic pathways is provided.

The Dual Role of HIF Hydroxylase PHD3 in Cancer Cell Survival

Most advanced tumours face periods of reduced oxygen availability i.e. hypoxia. During these periods tumour cells undergo adaptive changes enabling their survival under adverse conditions. In cancer hypoxia-induced cellular changes cause tumour progression, hinder cancer treatment and are indicative of poor prognosis. Within cells the main regulator of hypoxic responses is the hypoxia-inducible factor (HIF). HIF governs the expression of over a hundred hypoxia-inducible genes that regulate a number of cellular functions such as angiogenesis, glucose metabolism and cell migration. Therefore the activity of HIF must be tightly governed. HIF is regulated by a family of prolyl hydroxylase enzymes, PHDs, which mark HIF for destruction in normoxia. Under hypoxic conditions PHDs lose much of their enzymatic activity as they need molecular oxygen as a cofactor. Out of the three PHDs (PHD1, 2 and 3) PHD2 has been considered to be the main HIF-1 regulator in normoxic conditions. PHD3 on the other hand shows the most robust induction in response to oxygen deprivation and it has been implied as the main HIF-1 regulator under prolonged hypoxia. SQSTM1/p62 (p62) is an adaptor protein that functions through its binding motifs to bring together proteins in order to regulate signal transduction. In non-stressed situations p62 levels are kept low but its expression has been reported to be upregulated in many cancers. It has a definitive role as an autophagy receptor and as such it serves a key function in cancer cell survival decisions. In my thesis work I evaluated the significance of PHD3 in cancer cell and tumour biology. My results revealed that PHD3 has a dual role in cancer cell fate. First, I demonstrated that PHD3 forms subcellular protein aggregates in oxygenated carcinoma cells and that this aggregation promotes apoptosis induction in a subset of cancer cells. In these aggregates an adaptor protein SQSTM1/p62 interacts with PHD3 and in so doing regulates PHD3 expression. SQSTM1/p62 expression is needed to keep PHD3 levels low in normoxic conditions. Its levels rapidly decrease in response to hypoxia allowing PHD3 protein levels to be upregulated and the protein to be diffusely expressed throughout the cell. The interaction between PHD3 and SQSTM1/p62 limits the ability of PHD3 to function on its hydroxylation target protein HIF-1alpha. Second, the results indicate that when PHD3 is upregulated under hypoxia it protects cancer cells by allowing cell cycle to proceed from G1 to S-phase. My data demonstrates that PHD3 may either cause cell death or protect the cells depending on its expression pattern and the oxygen availability of tumours.

Gene expression promoted by the SV40 DNA targeting sequence and the hypoxia-responsive element under normoxia and hypoxia

The main objective of the present study was to find suitable DNA-targeting sequences (DTS) for the construction of plasmid vectors to be used to treat ischemic diseases. The well-known Simian virus 40 nuclear DTS (SV40-DTS) and hypoxia-responsive element (HRE) sequences were used to construct plasmid vectors to express the human vascular endothelial growth factor gene (hVEGF). The rate of plasmid nuclear transport and consequent gene expression under normoxia (20% O2) and hypoxia (less than 5% O2) were determined. Plasmids containing the SV40-DTS or HRE sequences were constructed and used to transfect the A293T cell line (a human embryonic kidney cell line) in vitro and mouse skeletal muscle cells in vivo. Plasmid transport to the nucleus was monitored by real-time PCR, and the expression level of the hVEGF gene was measured by ELISA. The in vitro nuclear transport efficiency of the SV40-DTS plasmid was about 50% lower under hypoxia, while the HRE plasmid was about 50% higher under hypoxia. Quantitation of reporter gene expression in vitro and in vivo, under hypoxia and normoxia, confirmed that the SV40-DTS plasmid functioned better under normoxia, while the HRE plasmid was superior under hypoxia. These results indicate that the efficiency of gene expression by plasmids containing DNA binding sequences is affected by the concentration of oxygen in the medium.

The effect of hypoxia and reoxygenation in the response of mesangial cells to angiotensin II in vitro

INTRODUCTION: Mesangial cells (MC) may be involved in the glomerular alterations induced by ischemia/reperfusion injury. OBJECTIVE: To evaluate the response of immortalized MC (IMC) to 30 minutes of hypoxia followed by reoxygenation periods of 30 minutes (H/R30) or 24 hours (H/R24). METHODS: The intracellular calcium concentration ([Ca+2]i) was measured before (baseline) and after adding angiotensin II (AII, 10-5 M) in the presence and absence of glybenclamide (K ATP channel blocker). We estimated the level of intracellular ATP, nitric oxide (NO) and PGE2. RESULTS: ATP concentration decreased after hypoxia and increased after reoxygenation. Hypoxia and H/R induced increases in basal [Ca+2]i. AII induced increases in [Ca+2]i in normoxia (97 ± 9%), hypoxia (72 ± 10%) or HR30 (85 ± 17%) groups, but there was a decrease in the response to AII in group H/R24 since the elevation in [Ca+2]i was significantly lower than in control (61 ± 10%, p < 0.05). Glybenclamide did not modify this response. It was observed a significant increase in NO generation after 24 hours of reoxygenation, but no difference in PGE2 production was observed. Data suggest that H/R injury is characterized by increased basal [Ca+2]i and by an impairment in the response of cells to AII. Results suggest that the relative insensibility to AII may be at least in part mediated by NO but not by prostaglandins or vasodilator K ATP channels. CONCLUSION: H/R caused dysfunction in IMC characterized by increases in basal [Ca+2]i during hypoxia and reduction in the functional response to AII during reoxygenation.

Modulation of TGF-β signaling by Hypoxia

A tumor is a fast-growing malignant tissue. This creates areas inside the tumor that are distant from local blood vessels to be able to get enough oxygen. This hypoxic condition activates a transcription factor called hypoxia inducible factor (HIF). HIF responses help a cell to adapt to decreased oxygen by activating glycolytic and angiogenesis pathways and by regulating apoptotic responses. Hypoxia drives the upregulation of a growth factor called transforming growth factor beta (TGF-beta). Similar to a hypoxia response, TGF is an important regulator of cell fate. TGF-β and HIF pathways regulate partially overlapping target genes. This regulation can also be cooperative. The TGF-beta signal is initiated by activation of plasma membrane receptors that then activate effector proteins called small mothers against decapentaplegic (Smad) homologs. In healthy tissue, TGF-β keeps cell proliferation and growth under control. During cancer progression, TGF-beta has shown a dual role, whereby it inhibits initial tumor formation but, conversely, in an existent tumor, TGF-beta drives malignant progression. Along with HIF and TGF-beta also protein dephosphorylation is an important regulatory mechanism of cell fate. Protein dephosphorylation is catalyzed by protein phosphatases such as Protein phosphatase 2A (PP2A). PP2A is a ubiquitous phosphatase that can exist in various active forms. PP2A can specifically regulate TGF-beta signaling either by enhancing or inhibiting the receptor activity. This work demonstrates that during hypoxia, PP2A is able to fine-tune TGF-beta signal by specifically targeting Smad3 effector in a Smad7-dependent manner. Inactivation of Smad3 in hypoxia leads to malignant conversion of TGF-beta signaling.

Life without Phd? – Phd Oxygen Sensor Proteins in Hypoxic Carcinoma Cell Survival

The microenvironment within the tumor plays a central role in cellular signaling. Rapidly proliferating cancer cells need building blocks for structures as well as nutrients and oxygen for energy production. In normal tissue, the vasculature effectively transports oxygen, nutrient and waste products, and maintains physiological pH. Within a tumor however, the vasculature is rarely sufficient for the needs of tumor cells. This causes the tumor to suffer from lack of oxygen (hypoxia) and nutrients as well as acidification, as the glycolytic end product lactate is accumulated. Cancer cells harbor mutations enabling survival in the rough microenvironment. One of the best characterized mutations is the inactivation of the von Hippel-Lindau protein (pVHL) in clear cell renal cell carcinoma (ccRCC). Inactivation causes constitutive activation of hypoxia-inducible factor HIF which is an important survival factor regulating glycolysis, neovascularization and apoptosis. HIFs are normally regulated by HIF prolyl hydroxylases (PHDs), which in the presence of oxygen target HIF α-subunit to ubiquitination by pVHL and degradation by proteasomes. In my thesis work, I studied the role of PHDs in the survival of carcinoma cells in hypoxia. My work revealed an essential role of PHD1 and PHD3 in cell cycle regulation through two cyclin-dependent kinase inhibitors (CKIs) p21 and p27. Depletion of PHD1 or PHD3 caused a cell cycle arrest and subjected the carcinoma cells to stress and impaired the survival.

The role of transcription factor Pitx1 and its regulation by hypoxia in Adolescent Idiopathic Scoliosis

La scoliose idiopathique de l’adolescent (SIA) est définie comme une courbure de la colonne vertébrale supérieure à 10 degrés, qui est de cause inconnue et qui affecte de façon prépondérante les adolescents. Des études précédentes sur des modèles murins ont démontré une inactivation partielle du gène Pitx1. Cette inactivation partielle provoque une déformation spinale sévère lors du développement des souris Pitx1+/-, ce qui est grandement similaire au phénotype de la SIA. En se basant sur ces observations, nous postulons que la perte de fonction de Pitx1 pourrait avoir un rôle dans la SIA et pourrait être régulée par des mécanismes moléculaires spécifiques. En effet, des études faites sur l’expression de Pitx1 révèlent une perte de son expression dans les ostéoblastes dérivés de patients SIA au niveau de l’ARNm. Nous émettons l’hypothèse que la perte de Pitx1 dans la SIA pourrait être déclenchée par des facteurs hypoxiques puisqu’il est connu que Pitx1 est réprimé par l’hypoxie et que HIF-2 alpha est surexprimés dans les ostéoblastes des patients SIA même dans des conditions normoxiques. De plus, nous avons découvert une mutation dans le domaine ODD des HIF-1 alpha chez certains patients SIA (3,1%). Une fonction connue de ce domaine est de stabiliser et d’augmenter l’activité transcriptionnelle de HIF-1 alpha dans des conditions normoxiques. Nous avons confirmé, par la technique EMSA, l’existence d’un élément de réponse fonctionnel à l’hypoxie au niveau du promoteur de Pitx1. Cependant, des co-transfections avec des vecteurs d’expression pour HIF-1 alpha et HIF-2 alpha, en présence de leur sous-unité beta ARNT, ont conduit à une activation du promoteur de Pitx1 dans la lignée cellulaire MG-63 ainsi que dans les ostéoblastes des sujets contrôles. Il est intéressant de constater qu’aucune activité du promoteur de Pitx1 dans les ostéoblastes SIA n’a été observée, même après la co-expression de HIF-2 alpha et ARNT, confirmant le fait que l’expression de Pitx1 est abrogée dans la SIA. Dans l’ensemble, nos résultats démontrent un rôle important de Pitx1 dans la SIA et une possible régulation par des facteurs hypoxiques.

Optimization Studies to Improve MSC-based Cardiac Cell Therapy : Cytokine Preconditioning and Nanoparticle Coupling

Contexte: La cardiopathie ischémique (IHD) reste une cause majeure de mortalité en Amérique du Nord. La thérapie cellulaire cardiaque (CCT) a émergé comme une thérapie prometteuse pour aider à guérir certaines malades cardiaques. Parmi les cellulaires avec propriétés pluripotentes, les cellules stromales mésenchymateuses (MSC) sont prometteuses. Cependant, plusieurs questions demeurent non résolues et certaines défis empêchent l'application clinique de la CCT se dans l'IHD, tels que le faible taux de rétention cellulaire in situ, le suivi des cellules in vivo post-implantation et post-acheminements et l`apoptose. Ici, le traitement préliminaire des MSC avec des facteurs de croissance et leur couplage avec des nanoparticules (NP) seront étudiés comme des méthodes pour optimiser MSC. Méthodes: Des MSCs provenant du rat (rMSC) et du cochon (pMSC) ont été isolés à partir de moelle osseuse. Les rMSC ont été préconditionnées avec SDF-1a, TSG-6 et PDGF-BB, et ensuite soumises à une hypoxie, une privation de sérum et a un stress oxydatif. Des études de cicatrisation ont également été effectués avec rMSCs préconditionnées. En parallèle, de nouvelles NP ferromagnétiques liées aux silicones ont été synthétisées. Les NPs ont été couplées aux pMSCs suivant leur fonctionnalisation avec l`anticorps, CD44, un antigène de surface du MSC bien connu. Par la suite, les études de biocompatibilité ont été réalisées sur pMSC-NP et en incluant des tests des processus cellulaires tels que la migration, l'adhésion, la prolifération et les propriétés de la différenciation. Résultats: Parmi toutes les cytokines testées, PDGF-BB a démontré la plus grande capacité à améliorer la survie de MSC dans des conditions d'hypoxie, de privation de sérum et en reponse au stress oxydatif. La conjugaison de NP a atténué la migration et la prolifération des pMSCs, mais n`a pas changé leur capacité de différenciation. Enfin, la complexe du MSC-NP est détectable par IRM. Conclusion: Nos données suggèrent que de nouvelles stratégies, telles que traitement préliminaire de PDGF-BB et le couplage des nanoparticules ferromagnétiques, peuvent être considérés comme des avenues prometteuse pour optimiser les MSCs pour la CCT.

Dynamic behavior of Arabidopsis eif4a-iii, putative core protein of exon junction complex: fast relocation to nucleolus and splicing speckles under hypoxia

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

Hypoxia and neurodegeneration

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimer's disease (AD). AD is characterized in part by the increased production of amyloid beta peptide (Abeta), which forms the extracellular plaques by which the disease can be identified post mortem. Numerous studies have now shown that hypoxia, even in vitro, can increase production of Abeta in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Abeta precursor, APP, and also the expression of the secretase enzymes, which cleave Abeta from APP. Other studies implicate reduced Abeta degradation as a possible means by which hypoxia increases Abeta levels. Such variability may be attributable to cell-specific responses to hypoxia. Further evidence indicates that some, but not all of the cellular adaptations to chronic hypoxia (including alteration of Ca(2+) homeostasis) require Abeta formation. However, other aspects of hypoxic remodeling of cell function appear to occur independently of this process. The molecular and cellular responses to hypoxia contribute to our understanding of the clinical association of hypoxia and increased incidence of AD. However, it remains to be determined whether inhibition of one or more of the effects of hypoxia may be of benefit in arresting the development of this neurodegenerative disease.

Hypoxia suppresses glutamate transport in astrocytes

Glutamate uptake by astrocytes is fundamentally important in the regulation of CNS function. Disruption of uptake can lead to excitotoxicity and is implicated in various neurodegenerative processes as well as a consequence of hypoxic/ischemic events. Here, we investigate the effect of hypoxia on activity and expression of the key glutamate transporters excitatory amino acid transporter 1 (EAAT1) [GLAST (glutamate-aspartate transporter)] and EAAT2 [GLT-1 (glutamate transporter 1)]. Electrogenic, Na+-dependent glutamate uptake was monitored via whole-cell patch-clamp recordings from cortical astrocytes. Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake was significantly reduced, and pharmacological separation of uptake transporter subtypes suggested that the EAAT2 subtype was preferentially reduced relative to the EAAT1. This suppression was confirmed at the level of EAAT protein expression (via Western blots) and mRNA levels (via real-time PCR). These effects of hypoxia to inhibit glutamate uptake current and EAAT protein levels were not replicated by desferrioxamine, cobalt, FG0041, or FG4496, agents known to mimic effects of hypoxia mediated via the transcriptional regulator, hypoxia-inducible factor (HIF). Furthermore, the effects of hypoxia were not prevented by topotecan, which prevents HIF accumulation. In stark contrast, inhibition of nuclear factor-kappaB (NF-kappaB) with SN50 fully prevented the effects of hypoxia on glutamate uptake and EAAT expression. Our results indicate that prolonged hypoxia can suppress glutamate uptake in astrocytes and that this effect requires activation of NF-kappaB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

Effect of hypobaric hypoxia, simulating conditions during long-haul air travel, on coagulation, fibrinolysis, platelet function, and endothelial activation

CONTEXT: The link between long-haul air travel and venous thromboembolism is the subject of continuing debate. It remains unclear whether the reduced cabin pressure and oxygen tension in the airplane cabin create an increased risk compared with seated immobility at ground level. OBJECTIVE: To determine whether hypobaric hypoxia, which may be encountered during air travel, activates hemostasis. DESIGN, SETTING, AND PARTICIPANTS: A single-blind, crossover study, performed in a hypobaric chamber, to assess the effect of an 8-hour seated exposure to hypobaric hypoxia on hemostasis in 73 healthy volunteers, which was conducted in the United Kingdom from September 2003 to November 2005. Participants were screened for factor V Leiden G1691A and prothrombin G20210A mutation and were excluded if they tested positive. Blood was drawn before and after exposure to assess activation of hemostasis. INTERVENTIONS: Individuals were exposed alternately (> or =1 week apart) to hypobaric hypoxia, similar to the conditions of reduced cabin pressure during commercial air travel (equivalent to atmospheric pressure at an altitude of 2438 m), and normobaric normoxia (control condition; equivalent to atmospheric conditions at ground level, circa 70 m above sea level). MAIN OUTCOME MEASURES: Comparative changes in markers of coagulation activation, fibrinolysis, platelet activation, and endothelial cell activation. RESULTS: Changes were observed in some hemostatic markers during the normobaric exposure, attributed to prolonged sitting and circadian variation. However, there were no significant differences between the changes in the hypobaric and the normobaric exposures. For example, the median difference in change between the hypobaric and normobaric exposure was 0 ng/mL for thrombin-antithrombin complex (95% CI, -0.30 to 0.30 ng/mL); -0.02 [corrected] nmol/L for prothrombin fragment 1 + 2 (95% CI, -0.03 to 0.01 nmol/L); 1.38 ng/mL for D-dimer (95% CI, -3.63 to 9.72 ng/mL); and -2.00% for endogenous thrombin potential (95% CI, -4.00% to 1.00%). CONCLUSION: Our findings do not support the hypothesis that hypobaric hypoxia, of the degree that might be encountered during long-haul air travel, is associated with prothrombotic alterations in the hemostatic system in healthy individuals at low risk of venous thromboembolism.