Hypoxia

Abstract Radiation therapy is a standard type of treatment modality used to achieve local control in more than 50% of all cancer patients. However, tumor hypoxia reduces the effectiveness of radiation therapy by limiting the biologically effective dose. We identified that FDA-approved phosphodiesterase 10A (PDE10A) inhibitor papaverine (PPV) has an off-target effect inhibiting mitochondrial oxygen consumption (OCR) leading to acute reduction of tumor hypoxia and significant radiosensitization of model tumors. Our data suggests that PDE10A does not contribute to the radiosensitizing effect. Based on PPV’s structure, we used biomedicinal chemistry approach to engineer its novel derivative SMV-32 with reduced PDE10A inhibition. We used Seahorse metabolic flux analyzer to determine the effect of PPV and SMV-32 on mitochondrial OCR in vitro. Near-infrared optical spectroscopy and pODD-luciferase in vivo reporter system was used for pharmacodynamics analyses and dose response evaluation. Local radiation therapy was delivered using Small Animal Radiation Research Platform (SARRP). We compared intraperitoneal (IP) and intravenous (IV) delivery and optimized dosing using pODD luciferase reporter system. SMV-32 showed superior OCR inhibition in vitro and enhanced hypoxia reduction in vivo compared to PPV. A single clinically relevant dose of PPV or SMV-32 significantly reduced the hypoxic fractions of heterotopic and orthotopic tumors in mice and sensitized the tumors to radiation therapy. PPV and SMV-32 appear to be ideal candidates for clinical radiosensitization. PPV is already in phase I clinical trial for treatment of non-small cell lung cancer combined with stereotactic body radiation therapy. Citation Format: Martin Benej, Jinghai Wu, McKenzie Kreamer, Sandip Vibhute, Ioanna Papandreou, Nicholas C. Denko. Papaverine derivative smv-32 alleviates tumor hypoxia and radiosensitizes tumors by inhibiting mitochondrial metabolism [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-033.

Tumour hypoxia is associated with poor patient prognosis and therapy resistance. A unique transcriptional response is initiated by hypoxia which includes the rapid activation of numerous transcription factors in a background of reduced global transcription. Here, we show that the biological response to hypoxia includes the accumulation of R-loops and the induction of the RNA/DNA helicase SETX. In the absence of hypoxia-induced SETX, R-loop levels increase, DNA damage accumulates, and DNA replication rates decrease. Therefore, suggesting that, SETX plays a role in protecting cells from DNA damage induced during transcription in hypoxia. Importantly, we propose that the mechanism of SETX induction in hypoxia is reliant on the PERK/ATF4 arm of the unfolded protein response. These data not only highlight the unique cellular response to hypoxia, which includes both a replication stress-dependent DNA damage response and an unfolded protein response but uncover a novel link between these two distinct pathways.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an almost universally fatal malignancy and there is an urgent need for new therapeutic targets. PDAC cells harbor genetic alterations and display metabolic changes that render them vulnerable to perturbations in redox homeostasis. Peroxiredoxin 4 (PRDX4) supports redox homeostasis by metabolizing H2O2 in the endoplasmic reticulum (ER). Based on functional genomics, we found that PDAC cell lines are dependent on PRDX4 for their growth and survival. We validated this dependency in established and primary PDAC cells, as well as in 3D models and orthotopic xenografts. PRDX4 depletion led to increased levels of reactive oxygen species (ROS). Tumor cells are exposed to fluctuating oxygen levels, which are known to augment ROS production. In line with this, PRDX4 addiction was also exacerbated in cells exposed to hypoxia and reoxygenation. Cell death induced by PRDX4 depletion was accompanied by DNA damage and a DNA-PKcs-governed DNA damage response. As such, PRDX4 depletion also sensitized cells and tumors to ionizing radiation. The source of ROS that created a dependency on PRDX4 was attributed to NADPH oxidase 4 (NOX4), which localizes to the ER membrane. The functional requirement for PRDX4 was correlated with cellular NADPH levels across different models of PDAC and could be rescued by depletion of NOX4 or NADPH. As such, this study has identified NOX4 as a link between metabolic deregulation and ER-specific redox vulnerability in PDAC. Since PRDX4 is not an essential gene in normal tissues, our work also suggests that PRDX4 represents a novel therapeutic target for pancreatic cancer that may be particularly potent in combination with radiotherapy. Citation Format: Marianne Koritzinsky, Pallavi Jain, Anna Dvorkin-Gheva, Lucie Malbeteau, Piriththiv Dhavarasa, Kevin R. Brown, Gun Ho Jang, Parth Vora, Faiyaz Notta, Jason Moffat, Paul C. Boutros. Metabolic deregulation drives a redox vulnerability in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-016.

BACKGROUND:Inflammation results in significant shifts in tissue metabolism. Recent studies indicate that inflammation and hypoxia occur concomitantly. We examined whether circulating and tissue markers of hypoxia could serve as surrogate indicators of disease severity in adult and pediatric patients with inflammatory bowel disease (IBD).METHODS:Serum and colonic biopsies were obtained from pediatric subjects with active IBD colitis and adult subjects with active and inactive ulcerative colitis, along with healthy non-colitis controls of all ages. Disease activity was evaluated by endoscopy and histopathology. Levels of serum hypoxia markers (macrophage inflammatory protein-3α [MIP-3α], vascular endothelial growth factor [VEGF], and erythropoietin [EPO]) were measured.RESULTS:Children with active IBD colitis had higher levels of serum MIP-3α and VEGF compared to non-colitis controls (p<0.01 and p<0.05, respectively). In adult subjects with endoscopically active ulcerative colitis, serum MIP-3α and EPO were significantly elevated compared to non-colitis controls (both p<0.01). In parallel, analysis of colon tissue MIP-3α mRNA and protein in pediatric subjects revealed increased expression in those with IBD colitis compared to controls (p<0.05 and p<0.01 for mRNA and protein, respectively). Serum MIP-3α and VEGF significantly increased with histology grade.CONCLUSION:Peripheral blood hypoxia markers may be useful indicators of disease activity for pediatric and adult IBD patients.

Hypoxia and its key mediators hypoxia inducible Factors (HIFs) are implicated in the development of liver diseases of diverse etiologies, often in interplay with inflammatory mediators. We investigated the interplay between hypoxia and proinflammatory mediators in the development of liver fibrosis, using human hepatocellular carcinoma Huh7 cells as a model. Treatment of Huh7 with DMOG or under hypoxia, induced HIF-1α protein levels and the expression of genes for pro-fibrotic (TGF-β1, PDGFC, PAI-1) and fibrosis (LOX, P4HA1, P4HB) markers. Knockdown of HIF-1α decreased the induction of PDGFC, LOX and P4HA1, showing the involvement of HIF-1 in their regulation. Interestingly, incubation of Huh7 cells under hypoxia did not cause activation of the NF-κΒ pathway. In contrast, inflammatory mediators such as tumor necrosis factor α (TNFα) and lipopolysaccharides (LPS) activated the NF-κΒ pathway, but failed to increase HIF-1α protein levels. Moreover, TNFα had a weaker effect than hypoxia on the induction or did not induce pro-fibrotic and fibrosis markers, respectively, while LPS enhanced only the hypoxic induction of P4HB. In conclusion, the above findings suggest that hypoxia and HIF-1 play an important role in the development of fibrosis in hepatocellular carcinoma, which appears to be independent of the activation of the NF-κΒ pathway.

Background: Loss of function mutations in the EGLN1 gene are a cause of erythrocytosis. EGLN1 encodes for prolyl hydroxylase domain protein 2 (PHD2). PHD2 hydroxylates and downregulates hypoxia-inducible factor-2 alpha (HIF-2 alpha), a transcription factor that regulates erythropoiesis. While the large majority of erythrocytosis-associated EGLN1 mutations occur within its catalytic domain, rare mutations reside in its zinc finger. This zinc finger binds a Pro-Xaa-Leu-Glu motif in p23, an HSP90 cochaperone that facilitates hydroxylation of HIF-alpha, an HSP90 client. Essentially nothing is known about the specific interactions between the PHD2 zinc finger and p23. Results: Here, we characterize an erythrocytosis-associated mutation in the zinc finger, K55N, that abolishes interaction with p23. We provide evidence that the affected residue, Lys-55, interacts with Asp-152 of p23. We also present results that indicate that PHD2 Arg-32 interacts with p23 Glu-160. Conclusion: These studies not only reinforce the importance of the PHD2 zinc finger in the control of erythropoiesis, but also lead to a model in which a peptide motif in p23 binds in a specific orientation to a predicted groove in the zinc finger of PHD2.

Purpose Nonalcoholic fatty liver disease (NAFLD) is considered the most common form of silent liver disease in the United States and obesity is associated with increased risk of NAFLD. Obstructive sleep apnea (OSA) which is common in obese individuals is associated with a greater incidence of NAFLD, which in turn, increases the risk for hepatocellular carcinoma (HCC). It is unclear how obesity, OSA and NAFLD interrelate nor how they collectively contribute to an increased risk for developing HCC. Patients and methods Male BALB/c mice were exposed to diethylnitrosamine and phenobarbital followed by 48 weeks of either standard chow diet (chow), chow with hypoxia, high-fat diet, or a combination of hypoxia and high-fat diet. We noninvasively monitored tumor development using micro-CT imaging. We tracked the total weight gained throughout the study. We evaluated liver histology, fat accumulation, carbonic anhydrase 9 (CA9) and hypoxia-inducible factor 1-alpha (HIF-1α) expression, as well as, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Results A high-fat diet without hypoxia led to the development of obesity that induced hepatic steatosis and promoted tumorigenesis. Animals on a high-fat diet and that were also exposed to hypoxia had lower total weight gain, lower steatosis, lower serum AST and ALT levels, and fewer number of hepatic adenomas than a high-fat diet without hypoxia. Conclusion These findings suggest that hypoxia abrogates obesity, hepatic steatosis, and hepatic tumorigenesis related to a high-fat diet.

Following a prolonged coronary arterial occlusion, heterogeneously scattered, focal regions of low erythrocyte flow are commonly found throughout the reperfused myocardium. Experimental studies have also demonstrated the presence of widespread, focally patchy regions of microvascular ischemia during reperfusion (RMI). However, the potential contribution of RMI to tissue viability and function has received little attention in the absence of practical clinical methods for its detection. In this review, the anatomic/functional basis of RMI is summarized, along with the evidence for its presence in reperfused myocardium. Advances in microcirculation research related to obstructive responses of vascular endothelial cells and blood elements to the effects of hypoxia and low shear stress are discussed, and a potential cycle of intensification of RMI from such responses and progressive loss of functional capillary density is presented. In capillaries with impaired erythrocyte flow, compensatory increases in the delivery of oxygen, because of its low solubility in plasma, are effective only at high partial pressures. As discussed herein, attenuation of the cycle with oxygen at hyperbaric levels in plasma is, very likely, responsible for improved tissue level perfusion noted experimentally. Observed clinical benefits from intracoronary SuperSaturated oxygen (SSO2) delivery, including infarct size reduction, can be attributed to attenuation of RMI with improvement in microvascular blood flow.

Purpose Bone marrow response to an organismal stress is made by orchestrating the interplay between hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Neither the cellular nor the molecular factors that regulate this process are fully understood, especially since this mechanism probably varies depending on the type of stress. Herein, we explored the differentiation and fate of MSCs and HSPCs in mice challenged with a hematopoietic stress or a mechanical stress applied separately or in combination. Methods Mice were subjected to 4 days of hypobaric hypoxia (hematopoietic challenge) and/or 7 days of hindlimb suspension (stromal challenge) and then sacrificed for blood and bone collection. Using hematological measurements, colony-forming unit assays, bone histomorphometry and array-based multiplex ELISA analysis, we evaluated challenge influences on both MSC and HSPC mobilization, differentiation (osteoblasts, osteoclasts, and mature blood cells) and fate. Results We found that hypoxia leads to HSPC mobilization and that an imbalance between bone formation and bone resorption accounts for this mobilization. Whilst suspension is also associated with an imbalance between bone formation and bone resorption, it does not induce HSPC mobilization. Then, we revealed cellular interactions by combining hematopoietic and stromal challenges together in mice. We showed that the hypoxia-driven HSPC mobilization is moderated by suspension. Moreover, when applied in a hypoxic environment, suspension offsets bone imbalance. We identified stroma cell-derived factors MIP-1α, HGF and SDF-1 as potent molecular key players sustaining interactions between hindlimb suspension and hypobaric hypoxia. Conclusion Taken together, our data highlight the benefit of combining different types of stress to better understand the interplay between MSCs and HSPCs.

Type 2 diabetes is caused by persistent high blood glucose, which is known as diabetic hyperglycemia. This hyperglycemic situation, when not controlled, can overproduce NADH and lower nicotinamide adenine dinucleotide (NAD), thereby creating NADH/NAD redox imbalance and leading to cellular pseudohypoxia. In this review, we discussed two major enzymatic systems that are activated by diabetic hyperglycemia and are involved in creation of this pseudohypoxic condition. One system is aldose reductase in the polyol pathway, and the other is poly (ADP ribose) polymerase. While aldose reductase drives overproduction of NADH, PARP could in contrast deplete NAD. Therefore, activation of the two pathways underlies the major mechanisms of NADH/NAD redox imbalance and diabetic pseudohypoxia. Consequently, reductive stress occurs, followed by oxidative stress and eventual cell death and tissue dysfunction. Additionally, fructose formed in the polyol pathway can also cause metabolic syndrome such as hypertension and nonalcoholic fatty liver disease. Moreover, pseudohypoxia can also lower sirtuin protein contents and induce protein acetylation which can impair protein function. Finally, we discussed the possibility of using nicotinamide riboside, an NAD precursor, as a promising therapeutic agent for restoring NADH/NAD redox balance and for preventing the occurrence of diabetic pseudohypoxia.

Purpose: Protein levels and activity of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are controlled by hydroxylation of the regulatory alpha chains.Proline hydroxylases (PHDs) target the protein for degradation via the von-Hippel-Lindau (VHL)ubiquitin-ligase complex, and asparagine hydroxylation by Factor Inhibiting HIF (FIH) leads to transcriptional inactivation.In cell-free systems, these enzymes require ascorbate as a cofactor, and this is also inferred to be an intracellular requirement for effective hydroxylation.However, how intracellular concentrations of ascorbate affect hydroxylase activity is unknown.In this study, we investigated the modulation of the regulatory hydroxylases in cancer cells by intracellular ascorbate.Materials and methods: To facilitate this investigation, we used clear cell renal carcinoma cell lines that were VHL-proficient (Caki-1), with a normal hypoxic response, or VHLdefective (Caki-2 and 786-0), with uncontrolled accumulation of HIF-α chains.We monitored the effect of intracellular ascorbate on the hypoxia-induced accumulation of HIF-1α, HIF-2α and the expression of downstream HIF targets BNIP3, cyclin D1 and GLUT1.Changes in hydroxylation of the HIF-1α protein in response to ascorbate were also investigated in 786-0 cells gene-modified to express full-length HIF-1α (786-HIF1).Results: In VHL-proficient cells, hypoxia induced accumulation of HIF-1α and BNIP3 which was dampened in mild hypoxia by elevated intracellular ascorbate.Increased HIF-2α accumulation occurred only under severe hypoxia and this was not modified by ascorbate availability.In VHL-defective cells, ascorbate supplementation induced additional accumulation of HIF under hypoxic conditions and HIF pathway proteins were unchanged by oxygen supply.In 786-HIF1 cells, levels of hydroxylated HIF-1α were elevated in response to increasing intracellular ascorbate levels.Conclusion: Our data provide evidence that the hypoxic pathway can be modulated by increasing HIF hydroxylase activity via intracellular ascorbate availability.In VHL-defective cells, accumulation of HIF-alpha proteins is independent of hydroxylation and is unaffected by intracellular ascorbate levels.

Background: Aryl hydrocarbon receptor (AHR), commonly known as an environmental sensor involved in the metabolism and elimination of xenobiotic substances, is also an important modulator in the development and functioning of the immune system. AHR expression is varied in the T cell subsets with the highest expression in T-helper 17 and T regulatory cells. It has been reported that AHR can act as a tumor promoter or a tumor suppressor, depending on the tumor type. Methods: In an effort to understand the role played by AHR in tumor growth, the MC38 syngeneic colon carcinoma tumor model was used on C57BL/6 or ahr knockout (KO, -/-) mice with or without AHR antagonist (CH223191) treatment. Tumor sizes were measured, and biomarkers were quantified in tumor microenvironment and draining lymph nodes using flow cytometry. Enzyme-linked immunosorbent assay was used to determine the amount of cytokines in tumors. Results: In ahr deficient mice, MC38 tumors progress more rapidly than in wild-type mice, accompanied by an increase in tumor-associated macrophages and M2 macrophages and a decrease in CD8a positive cytotoxic lymphocytes. Analysis of cytokines in the tumor microenvironment reveals a pro-inflammatory phenotype. Similar changes were observed by pharmacologic blockade of the receptor using CH223191. Conclusion: AHR acts as a tumor suppressor in mice implanted with MC38 colon carcinoma cells as evidenced by either a blockade or deficiency of AHR.

Background: The brain is extensively vascularized, uses similar to 20% of the body's oxygen, and is highly sensitive to changes in oxygen. While synaptic plasticity and memory are impaired in healthy individuals by exposure to mild hypoxia, aged individuals appear to be even more sensitive. Aging is associated with progressive failure in pulmonary and cardiovascular systems, exposing the aged to both chronic and superimposed acute hypoxia. The HIF proteins, the "master regulators" of the cellular response to hypoxia, are robustly expressed in neurons and astrocytes. Astrocytes support neurons and synaptic plasticity via complex metabolic and trophic mechanisms. The activity of HIF proteins in the brain is diminished with aging, and the increased exposure to chronic and acute hypoxia with aging combined with diminished HIF activity may impair synaptic plasticity. Purpose: Herein, we test the hypothesis that astrocyte HIF supports synaptic plasticity and learning upon hypoxia. Materials and Methods: An Astrocyte-specific HIF loss-of-function model was employed, where knock-out of HIF-1 alpha or HIF-2 alpha in GFAP expressing cells was accomplished by cremediated recombination. Animals were tested for behavioral (open field and rotarod), learning (passive avoidance paradigm), and electrophysiological (long term potentiation) responses to mild hypoxic challenge. Results: In an astrocyte-specific HIF loss-of-function model followed by mild hypoxia, we identified that the depletion of HIF-2 alpha resulted in an impaired passive avoidance learning performance. This was accompanied by an attenuated response to induction in long-term potentiation (LTP), suggesting that the hippocampal circuitry was perturbed upon hypoxic exposure following HIF-2 alpha loss in astrocytes, and not due to hippocampal cell death. We investigated HIF-regulated trophic and metabolic target genes and found that they were not regulated by HIF-2 alpha, suggesting that these specific targets may not be involved in mediating the phenotypes observed. Conclusion: Together, these results point to a role for HIF-2 alpha in the astrocyte's regulatory role in synaptic plasticity and learning under hypoxia and suggest that even mild, acute hypoxic challenges can impair cognitive performance in the aged population who harbor impaired HIF function.

Background Hypoxia in tumors is associated with resistance towards various therapies including radiotherapy. In this study, we assessed if hypoxia in cancer spheres could be effectively reduced by adding etomoxir (a β-oxidation inhibitor) immediately after cell irradiation. Methods We employed cancer cells’ sphere model to target hypoxia. Confocal imaging was used to analyze hypoxia and expression of specific biomarkers in spheres following various treatments (radiation and/or etomoxir). Results Etomoxir (32.5 μM) treatment improved the radiation (2.5 Gy) efficacy against growth of lung adenocarcinoma H460 spheres. More importantly, radiation and etomoxir combination significantly reduced the hypoxic regions (pimonidazole+ areas) in H460 spheres compared to either treatment alone. Also, etomoxir and radiation combination treatment reduced the protein level of biomarkers for proliferation (Ki-67 and cyclin D1), stemness (CD44) and β-oxidation (CPT1A) in H460 spheres. We observed similar efficacy of etomoxir against growth of prostate cancer LNCaP cells’ spheres when combined with radiation. Further, radiation treatment strongly reduced the hypoxic regions (pimonidazole+ areas) in CPT1 knockdown LNCaP cells’ spheres. Conclusions Together, these results offer a unique approach to target hypoxia in solid tumors via combining etomoxir with radiation, thereby improving therapeutic efficacy.

Purpose Hypoxia induces pulmonary vasoconstriction with a subsequent increase of pulmonary artery pressure (PAP), which can result in pulmonary hypertension. Serelaxin has shown an increase of pulmonary hemodynamic parameters after serelaxin injection. We therefore investigated the response of pulmonary hemodynamic parameters after serelaxin administration in a clinically relevant model. Methods Six controls and six sheep that received 30 μg/kg serelaxin underwent right heart catheterization during a 12-minute hypoxia period (inhalation of 5% oxygen and 95% nitrogen) and subsequent reoxygenation. Systolic, diastolic, and mean values of both PAP (respectively, PAPs, PAPd, and PAPm) and pulmonary capillary wedge pressure (respectively, PCWPs, PCWPd, and PCWPm), blood gases, heart rate (HR), and both peripheral and pulmonary arterial oxygen saturation were obtained. Cardiac output (CO), stroke volume (SV), pulmonary vascular resistance (PVR), pulmonary arterial compliance (PAcompl), and systemic vascular resistance (SVR) were calculated. Results The key findings of the current study are that serelaxin prevents the rise of PAPs (p≤0.001), PAPm, PCWPm, PCWPs (p≤0.03), and PAPd (p≤0.05) during hypoxia, while it simultaneously increases CO and SV (p≤0.001). Similar courses of decreases of PAPm, PAPd, PAPs, CO, SVR (p≤0.001), and PCWPd (p≤0.03) as compared to hypoxic values were observed during reoxygenation. In direct comparison, the experimental groups differed during hypoxia in regard to HR, PAPm, PVR, and SVR (p≤0.03), and during reoxygenation in regard to HR (p≤0.001), PAPm, PAPs, PAPd, PVR, SVR (p≤0.03), and PCWPd (p≤0.05). Conclusion The findings of this study suggest that serelaxin treatment improves pulmonary hemodynamic parameters during acute hypoxia.

The formation of a functional cardiovascular system is an essential step in the early vertebrate embryo. Nevertheless, the effect of hypoxia on the developmental program of organisms was studied rarely. In particular, this holds true for vertebrate embryos that depend on a functional placenta for proper development and had not been studied in this respect due to the obvious limitation. We established a protocol to culture aquatic embryos, which enabled us to culture a high number of Xenopus embryos until tadpole stage under defined hypoxic conditions in four hypoxia chambers simultaneously, employing a computerized system. In general, our results show that hypoxia results in delayed development and, in particular, we could show that oxygen availability was most crucial during gastrulation and organogenesis (early tailbud) phases during embryonic development of Xenopus laevis.

BACKGROUND:In humans and other animals, the chronic hypoxic response is mediated by hypoxia inducible transcription factors (HIFs) which regulate the expression of genes that counteract the effects of limiting oxygen. Prolyl hydroxylases (PHDs) act as hypoxia sensors for the HIF system in organisms ranging from humans to the simplest animal Trichoplax adhaerens. METHODS:We report structural and biochemical studies on the T. adhaerens HIF prolyl hydroxylase (TaPHD) that inform about the evolution of hypoxia sensing in animals. RESULTS:High resolution crystal structures (≤1.3 Å) of TaPHD, with and without its HIFα substrate, reveal remarkable conservation of key active site elements between T. adhaerens and human PHDs, which also manifest in kinetic comparisons. CONCLUSION:Conserved structural features of TaPHD and human PHDs include those apparently enabling the slow binding/reaction of oxygen with the active site Fe(II), the formation of a stable 2-oxoglutarate complex, and a stereoelectronically promoted change in conformation of the hydroxylated proline-residue. Comparison of substrate selectivity between the human PHDs and TaPHD provides insights into the selectivity determinants of HIF binding by the PHDs, and into the evolution of the multiple HIFs and PHDs present in higher animals.

The "Hypoxia Nantes 2016" organized its second conference dedicated to the field of hypoxia research. This conference focused on "the role of hypoxia under physiological conditions as well as in cancer" and took place in Nantes, France, in October 6-7, 2016. The main objective of this conference was to bring together a large group of scientists from different spheres of hypoxia. Recent advances were presented and discussed around different topics: genomics, physiology, musculoskeletal, stem cells, microenvironment and cancer, and oxidative stress. This review summarizes the major highlights of the meeting.

Objective Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. Methods Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis. Results Exposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels. Conclusion Moderate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.