Hypoxic Response Research Articles

NIMG-26. GLIOBLASTOMA TISSUE INVASION MAPPING WITH NOVEL HYPOXIA-TARGETED BLOOD OXYGENATION-LEVEL DEPENDENT MRI

Abstract BACKGROUND Glioblastoma is a devastating incurable malignant primary brain tumor exhibiting an erratic growth pattern and brain tissue invasion exceeding the typically depicted contrast-enhancing lesion on MRI. This behaviour is strongly associated with the presence of aberrant vasculature. Advanced clinical neuroimaging techniques, however, have not provided convincing imaging markers to determine glioblastoma tissue infiltration and monitor treatment effects. We investigated a new imaging technique based on blood oxygenation-level dependent (BOLD) MRI contrast with precise hypoxic respiratory targeting in order to better determine tumor tissue invasion not restricted to the typically depicted focal glioblastoma lesion on contrast-enhanced MRI. METHODS A computer-controlled gas blender was used to induce transient and standardized hypoxic stimulus in isocapnic conditions during echo-planar-imaging acquisition in patients with brain tumors.8,9 Data were preprocessed using SPM and in-house written MATLAB scripts. The induced BOLD signal changes were used to calculate %BOLD signal change during hypoxic stimulus with respect to the baseline, contrast to noise (CNR) ratio and goodness-of-fit (Rsquared) for each voxel in the whole-brain and automatically segmented region of interest (contrast-enhancement, necrosis, edema) and overlayed using color-maps onto anatomical high resolution T1. RESULTS Hypoxic targeting during gas modulation could be performed with high reproducibility, caused no discomfort in patients with brain tumors and was able to induce strong signal change during hypoxic stimulus with high contrast-noise ratio and Rsquared. In particular, when compared with other tumors, BOLD hypoxia glioblastoma tissue invasion maps show very distinct hypoxic signal responses in some cases extending beyond the focal -contrast-enhancing- glioblastoma lesion. CONCLUSION The ability to map at voxel level BOLD hypoxia signal patterns may determine a novel therapeutic imaging marker based on structural vascular alterations in the tumour tissue infiltration zone before these become appreciable with standard neuroimaging. This may support a refinement and tailoring of tumor resection and improve follow-up after resection and during treatment i.e. chemoradiotherapy.

Transcriptomic and Metabolomic Insights into Age-Related Changes in Lung Tissue of Yaks Under Highland Stress

As an indigenous species on the Tibetan Plateau, the yak is well adapted to the plateau hypoxic environment. The high-altitude hypoxia adaptation of the yak requires the adaptive reshaping of multiple tissues and organs, especially the lungs. To reveal the adaptive development of yak lungs under hypoxic stress at the tissue and molecular levels, we conducted histomorphological observations as well as transcriptomic and metabolomic studies of yak lungs at three ages (0.5, 2.5, and 4.5 years). The results showed that the lung tissue developed significantly with age. The mean alveolar area was higher (p < 0.01) in 4.5 and 2.5-year-old yaks than in 0.5-year-old yaks. The percentage of elastic fibers, micro-arterial wall thickness, and micro-arterial area showed an increasing trend (p < 0.01) from 0.5-year-old yaks to 2.5-year-old yaks and then to 4.5-year-old yaks. In addition, some critical differentially expressed genes related to angiogenesis (MYC, EPHA2, TNF), fiber formation (EREG), smooth muscle proliferation (HBEGF), erythropoiesis (SOCS3), and hypoxia response (ZFP36) were identified. Some metabolites associated with these genes were also found simultaneously. These findings provide a deeper understanding of the molecular strategies underlying this species’ extraordinary ability to survive normally in low-oxygen environments. In conclusion, the lungs of yaks undergo continuous adaptive development under hypoxic stress, and these findings are crucial for understanding the molecular mechanisms by which native species of the Tibetan Plateau survive in harsh environments.

Identification of stimuli that enhance human herpesvirus 6A (HHV-6A) replication and reconstitution

ABSTRACT Despite the availability of bacterial artificial chromosome (BAC) systems for human herpesvirus 6A (HHV-6A), reconstitution of infectious viruses is very challenging and time consuming. In this study, we developed approaches to improve the reconstitution process and enhance virus replication to overcome these technical challenges. Using dimethyl sulfoxide and exonuclease V, we significantly increased the efficiency of BAC transfections into JJHan T cells. We tested several stimulation strategies to enhance lytic replication and identified mitogens and glucocorticoids that, in combination, improve virus replication. In addition, we demonstrated that the interferon-mediated response impairs virus reconstitution and that the JAK1/JAK2 inhibitor ruxolitinib resulted in an immense improvement. Furthermore, hypoxia-inducible factor 1 alpha stabilization by IOX2 drastically accelerated virus reconstitution, indicating that the hypoxic response is a crucial regulator of HHV-6A replication. Our study sheds light on strategic approaches that improve replication and reconstitution of this ubiquitous human herpesvirus. IMPORTANCE HHV-6A is a betaherpesvirus that infects a wide range of human tissues and establishes lifelong latency in the host. Its reactivation has been implicated in several diseases, including multiple sclerosis, encephalitis, myocarditis, and chronic fatigue syndrome, although its pathogenetic role remains elusive. The efficacy of common antiviral drugs is limited, and no specific drugs target HHV-6A infection. The data of this study shed light on stimuli and potential pathways that influence HHV-6A replication and reconstitution. Our strategies not only simplify virus propagation and reconstitution to study HHV-6A biology but also provide the basis for the development of therapeutic strategies.

Adipocytes reprogram the proteome of breast cancer cells in organotypic three-dimensional cell cultures.

While epidemiological evidence has long linked obesity with an increased risk of breast cancer, the intricate interactions between adipocytes and cancer cells within the tumor microenvironment remain largely uncharted territory. The use of organotypic three-dimensional (3D) cell cultures that more accurately mimic the spatial architecture of tumors represents an innovative approach to this complex issue. In the present study, we investigated the effects of adipocytes on the proteome of Hs578t breast cancer cells cultured in a 3D microenvironment. Using different treatments, we rigorously optimized the experimental conditions to induce the optimal differentiation of 3T3-L1 fibroblasts into mature adipocytes. Then, we grow the Hs578t cells in a simulated microenvironment using an on-top model for organotypic 3D cultures. Our data showed that cancer cells formed 3D stellate-like architectures when grown over an extracellular matrix proteins-enriched scaffold for 48h. Proteomic profiling using LC-MS/MS mass spectrometry of Hs578t cells grown in 3D conditions with or without the adipocyte-enriched culture discovered 916 unique proteins. Of these, 605 showed no significant changes in abundance, whereas 87 proteins were significantly upregulated and 224 downregulated after interaction with fat cells (p < 0.05, FC > 2.0). Bioinformatic analysis of upregulated proteins indicated that the most enriched GO terms and molecular functions were related to lipids transport, cell differentiation, hypoxia response, and cell junctions. In addition, several modulated proteins have been previously associated with breast cancer progression. Interestingly, lipid transport proteins, including PITPNM2, ATP2C1, ABCA12, HDLBP, and APOB, showed perturbations in their expression, which were also associated with low overall survival in breast cancer patients. Functional studies showed that the knockdown of apolipoprotein B (APOB) expression in Hs578t cells reduced the size of 3D cellular structures. Moreover, APOB-knocked cells cocultured with adipocytes for 48h exhibited a significant decrease of intracellular lipids, whereas an increase in the adipocytes was found. Our results indicate that the 3D microenvironment and the adipocytes crosstalk reprogram the proteome of breast cancer cells. These data help us understand the environmental effects in gene expression and contribute to discovering novel tumor proteins with potential intervention in breast cancer therapy.

The impact of early surgery on mortality and functional recovery in older adults with traumatic intracranial lesions: a propensity score-based analysis.

There is skepticism about the benefit of surgery in elderly patients affected by traumatic brain injury (TBI) due to the negative effect of age on the outcome and surgical complications. However, there are few studies that have investigated differences in patient's outcome between surgically and conservatively managed patients after adjusting for the imbalance in preinjury characteristics and clinical and radiological features. The primary aim of this study was to evaluate the effect of early surgery on mortality and functional recovery in a cohort of older adults with acute traumatic intracranial lesions after adjustment by Propensity Score (PS) matching. MATERIALS AND METHODS: We conducted a retrospective cohort study on older adult patients (≥ 65 years) admitted for TBI between 2013 and 2023 to a single level 1 trauma center. Patients were categorized based on whether they underwent early surgery (< 48h after TBI) for a space-occupying lesion evacuation. PS model was constructed based on age, frailty, comorbidities (Charlson comorbity index and American Society of Anaesthesiologists score), anticoagulants, hypoxia, shock, pupillary abnormalities and GCS motor response upon admission, midline shift, basal cistern effacement, volume of subdural and intracerebral hematomas, and limitation of life-sustaining treatment decisions.The effect of early surgery on 30-day mortality and unfavorable functional outcomes (GOSE 1-3) at 6 and 12 months were investigated after matching by paired test. We identified and reviewed 301 patients who met all inclusion criteria and contained no exclusions. After matching, 62 patients (31 pairs of conservative and surgical patients) remained as the matched datasets. Our key finding was that older adult TBI patients who underwent early surgery had a statistically significant reduction in the risk of 30-day mortality (OR 0.313, 95% CI 0.114-0.853, p = 0.023) and unfaourable outcome at 12 months after TBI (OR 0.286, 95% CI 0.094-0.868, p = 0.027). Early surgery was associated with decreased 30-day mortality and better functional outcome at 12 months after TBI in older adults with few comorbidities and good functionality when clinically affected by acute traumatic intracranial lesions with mass effect.

Respiratory Dysfunction and Abnormal Hypoxic Ventilatory Response in Mild to Moderate Parkinson's Disease.

Respiratory dysfunction is an important contributor to morbidity and mortality in advanced Parkinson's disease (PD), but it is unclear what parameters are sensitive to diagnose and monitor respiratory dysfunction across disease phases. We aimed to characterize respiratory dysfunction in mild to moderate PD. In 20 individuals without cardiopulmonary comorbidity, pulmonary and inspiratory muscle function testing were performed ON-medication. Subsequently, the acute ventilatory response to hypoxia (HVR) was assessed by gradually decreasing FIO2 from 0.209 (room air) to 0.127, which was compared to eight age- and sex-matched healthy controls under arterial blood gas monitoring. Lastly, on different days, the same 20 individuals with PD underwent six blinded exposures to 45-min normobaric hypoxia at FiO2 0.163 and 0.127 or placebo OFF-medication to assess breathing responses. At rest, individuals with greatest PD severity had a lower tidal volume (pairwise comparisons: 0.59 vs. 0.74, P = 0.038-0.050) and tended to have a higher breathing frequency (17.7 vs. 14.4, P = 0.076), despite normal pulmonary function. A 45-min exposure to hypoxia induced a significantly lower acute HVR in individuals with PD compared to controls (-0.0489 vs. 0.133 L.min/%, P = 0.0038). Acute HVR was reduced regardless of disease severity. Subacute HVR in individuals with milder disease tended to be higher compared to those with more advanced disease (P = 0.079). Respiratory dysfunction is present in individuals with PD, including those with relatively mild disease severity, and is characterized by altered breathing patterns at rest, as well as a lower HVR, despite normal pulmonary and inspiratory muscle function testing.

Elevated Perspectives: Unraveling Cardiovascular Dynamics in High-Altitude Realms.

High-altitude regions pose distinctive challenges for cardiovascular health because of decreased oxygen levels, reduced barometric pressure, and colder temperatures. Approximately 82 million people live above 2400 meters, while over 100 million people visit these heights annually. Individuals ascending rapidly or those with pre-existing cardiovascular conditions are particularly vulnerable to altitude-related illnesses, including Acute Mountain Sickness (AMS) and Chronic Mountain Sickness (CMS). The cardiovascular system struggles to adapt to hypoxic stress, which can lead to arrhythmias, systemic hypertension, and right ventricular failure. Pathophysiologically, high-altitude exposure triggers immediate increases in cardiac output and heart rate, often due to enhanced sympathetic activity. Over time, acclimatisation involves complex changes, such as reduced stroke volume and increased blood volume. The pulmonary vasculature also undergoes significant alterations, including hypoxic pulmonary vasoconstriction and vascular remodelling, contributing to conditions, like pulmonary hypertension and high-altitude pulmonary edema. Genetic adaptations in populations living at high altitudes, such as gene variations linked to hypoxia response, further influence these physiological processes. Regarding cardiovascular disease risk, stable coronary artery disease patients generally do not face significant adverse outcomes at altitudes up to 3500 meters. However, those with unstable angina or recent cardiac interventions should avoid high-altitude exposure to prevent exacerbation. Remarkably, high-altitude living correlates with reduced cardiovascular mortality rates, possibly due to improved air quality and hypoxia-induced adaptations. Additionally, there is a higher incidence of congenital heart disease among children born at high altitudes, highlighting the profound impact of hypoxia on heart development. Understanding these dynamics is crucial for managing risks and improving health outcomes in high-altitude environments.

Atypical chemokine receptor 2 expression is directly regulated by hypoxia inducible factor-1 alpha in cancer cells under hypoxia

Lack of significant and durable clinical benefit from anti-cancer immunotherapies is partly due to the failure of cytotoxic immune cells to infiltrate the tumor microenvironment. Immune infiltration is predominantly dependent on the chemokine network, which is regulated in part by chemokine and atypical chemokine receptors. We investigated the impact of hypoxia in the regulation of Atypical Chemokine Receptor 2 (ACKR2), which subsequently regulates major pro-inflammatory chemokines reported to drive cytotoxic immune cells into the tumor microenvironment. Our in silico analysis showed that both murine and human ACKR2 promoters contain hypoxia response element (HRE) motifs. Murine and human colorectal, melanoma, and breast cancer cells overexpressed ACKR2 under hypoxic conditions in a HIF-1α dependent manner; as such overexpression was abrogated in melanoma cells expressing non-functional deleted HIF-1α. We also showed that decreased expression of ACKR2 in HIF-1α-deleted cells under hypoxia was associated with increased CCL5 levels. Chromatin immunoprecipitation data confirmed that ACKR2 is directly regulated by HIF-1α at its promoter in B16-F10 melanoma cells. This study provides new key elements on how hypoxia can impair immune infiltration in the tumor microenvironment.

Hypoxic Responses in Periodontal Tissues: Influence of Smoking and Periodontitis.

This study aimed to investigate the hypoxic changes in periodontal tissues resulting from smoking and periodontitis by assessing levels of hypoxia-inducible factors (HIF-1α, HIF-2α, HIF-3α) and vascular endothelial growth factor (VEGF) in gingival crevicular fluid (GCF). The study comprised 22 periodontally healthy non-smokers (Group H), 22 periodontally healthy smokers (Group HS), 22 non-smokers with periodontitis (Group P) and 22 smokers with periodontitis (Group PS). Clinical periodontal parameters were documented, and GCF samples were collected and analysed using enzyme-linked immunosorbent assay (ELISA). Significantly elevated levels of HIF-1α, HIF-3α and VEGF were observed in Groups HS, P and PS compared to Group H (p < 0.05). Moreover, higher HIF-2α levels were detected in the Groups HS and P compared to Group H (p < 0.05). Significant correlations were detected between all evaluated hypoxia biomarkers in the Group P (p < 0.05) except HIF-2α and HIF-3α. However, in the PS group, significant correlation appeared only between HIF-1α and HIF-2α (p < 0.05). Our findings indicate that smoking and periodontitis induce comparable hypoxic effects in periodontal tissues, as evidenced by the evaluated biomarkers. Further research is warranted to gain a deeper understanding of the mechanisms underlying hypoxia in periodontal tissues.

Lineage Differentiation and Genomic Vulnerability in a Relict Tree From Subtropical Forests.

The subtropical forests of East Asia are renowned for their high plant diversity, particularly the abundance of ancient relict species. However, both the evolutionary history of these relict species and their capacity for resilience in the face of impending climatic changes remain unclear. Using whole-genome resequencing data, we investigated the lineage differentiation and demographic history of the relict and endangered tree, Bretschneidera sinensis (Akaniaceae). We employed a combination of population genomic and landscape genomic approaches to evaluate variation in mutation load and genomic offset, aiming to predict how different populations may respond to climate change. Our analysis revealed a profound genomic divergence between the East and West lineages, likely as the result of recurrent bottlenecks due to climatic fluctuations during the glacial period. Furthermore, we identified several genes potentially linked to growth characteristics and hypoxia response that had been subjected to positive selection during the lineage differentiation. Our assessment of genomic vulnerability uncovered a significantly higher mutation load and genomic offset in the edge populations of B. sinensis compared to their core counterparts. This implies that the edge populations are likely to experience the most significant impact from the predicted climate conditions. Overall, our research sheds light on the historical lineage differentiation and contemporary genomic vulnerability of B. sinensis. Broadening our understanding of the speciation history and future resilience of relict and endangered species such as B. sinensis, is crucial in developing effective conservation strategies in anticipation of future climatic changes.

Hypoxia-inducible factor-2α enhances neutrophil survival to promote cardiac injury following myocardial infarction.

Heart failure is a major cause of mortality following myocardial infarction. Neutrophils are among the first immune cells to accumulate in the infarcted region. Although beneficial functions of neutrophils in heart injury are now appreciated, neutrophils are also well known for their ability to exacerbate inflammation and promote tissue damage. Myocardial infarction induces hypoxia, where hypoxia-inducible factors (HIFs) are activated and play critical roles in cellular functions. In this context, the role of Hif2α in neutrophils during myocardial infarction is unknown. Here, we demonstrate that neutrophil Hif2α deletion markedly attenuates myocardial infarct size, improves cardiac function, reduces neutrophil survival and tissue accumulation, and correlates with increased macrophage engulfment rates. Mechanistic studies revealed that Hif2α promotes neutrophil survival through binding to hypoxia response element (HRE) in the promoter region of Birc2 to regulate expression of the prosurvival factor, cellular inhibitor of apoptosis protein-1 (cIAP1). Inhibition of cIAP1 in neutrophils using the pharmacological agent, Birinapant resulted in increased cell death, establishing a critical role of cIAP1 downstream of Hif2α in neutrophil survival. Taken together, our data demonstrate a protective effect of Hif2α deletion in neutrophils on cardiac injury outcomes through modulation of neutrophil cell survival.NEW & NOTEWORTHY Hif2α in neutrophils increases infarct size, cardiac dysfunction, and ventricular scar after myocardial infarction. Hif2α in neutrophils supports neutrophil survival via cIAP-1 signaling and delays macrophage engulfment.

Menstrual cycle does not impact the hypoxic ventilatory response and acute mountain sickness prediction

The relationship between the variations in ovarian hormones (i.e., estrogens and progesterone) and the hypoxic ventilatory response (HVR) remains unclear. HVR is a key adaptive mechanism to high altitude and has been proposed as a predictor for acute mountain sickness (AMS). This study aimed to explore the effects of hormonal changes across the menstrual cycle on HVR. Additionally, it assessed the predictive capacity of HVR for AMS and examined whether a particular menstrual phase could enhance its predictive accuracy. Thirteen eumenorrheic women performed a pure nitrogen breathing test near sea level, measuring HVR and cerebral oxygenation in early follicular, late follicular, and mid-luteal phases. Oxidative stress and ovarian hormone levels were also measured. AMS symptoms were evaluated after spending 14 h, including one overnight, at an altitude of 3,375 m. No differences in HVR, ventilation, peripheral oxygen saturation, or cerebral oxygenation were observed between the three menstrual cycle phases. Moreover, these parameters and the oxidative stress markers did not differ between the women with or without AMS (31% vs 69%), regardless of the menstrual cycle phase. In conclusion, ventilatory responses and cerebral oxygenation in normobaric hypoxia were consistent across the menstrual cycle. Furthermore, these parameters did not differentiate women with or without AMS.

Human prolyl hydroxylase domain 2 reacts with O2 and 2-oxoglutarate to enable formation of inactive Fe(III).2OG.hypoxia-inducible-factor α complexes

Hypoxia inducible transcription factors (HIFs) mediate the hypoxic response in metazoans. When sufficient O2 is present, Fe(II)/2-oxoglutarate (2OG)-dependent oxygenases (human PHD1-3) promote HIFα degradation via prolyl-hydroxylation. We report crystallographic, spectroscopic, and biochemical characterization of stable and inactive PHD2.Fe(III).2OG complexes. Aerobic incubation of PHD2 with Fe(II) and 2OG enables formation of PHD2.Fe(III).2OG complexes which bind HIF1-2α to give inactive PHD2.Fe(III).2OG.HIF1-2α complexes. The Fe(III) oxidation state in the inactive complexes was shown by EPR spectroscopy. L-Ascorbate hinders formation of the PHD2.Fe(III).2OG.(+/-HIFα) complexes and slowly regenerates them to give the catalytically active PHD2.Fe(II).2OG complex. Crystallographic comparison of the PHD2.Fe(III).2OG.HIF2α complex with the analogous anaerobic Fe(II) complex reveals near identical structures. Exposure of the anaerobic PHD2.Fe(II).2OG.HIF2α crystals to O2 enables in crystallo hydroxylation. The resulting PHD2.product structure, manifests conformational changes compared to the substrate structures. The results have implications for the role of the PHDs in hypoxia sensing and open new opportunities for inhibition of the PHDs and other 2OG dependent oxygenases by promoting formation of stable Fe(III) complexes.

MiRNA-519e promotes cardiomyocyte cell division accompanied by pro-angiogenic and anti-apoptotic effects

Abstract Background MicroRNAs have the therapeutic potential for heart repair after myocardial infarction(MI). Using a functional high-throughput screening approach, we identified microRNA-519e(miR-519e) as an inducer of cell-cycling in human-derived iPSC-cardiomyocytes(hiPSC-­CM). Purpose This study examines miR-519e for cell division in hiPSC-­CM, additional beneficial functions in heart-related non-CMs, and direct target identification and validation in vitro and in vivo. Methods MiR-519e-mimics or miR-scrambled(miR-scr) were transfected into hiPSC-­CM, human aortic endothelial cells(HAEC) and human cardiac fibroblasts(cFB). Proliferative markers (EdU(5-Ethynyl-2'- deoxyuridine), H3P(Phospho-Histone H3) and AURKB(Aurora B-kinase)) and apoptotic response using MTT assay after exposure to doxorubicin were assessed in hiPSC­-CMs. Angiogenic capacity of HAEC was evaluated by tube formation assay and fibrotic response of cFBs using EdU and α-SMA(smooth muscle actin) staining. In vivo, a mouse MI model with ligation of the left anterior descending followed by intramyocardial injection of miR-519e or miR-scr was used. Direct target identification of miR-519e was assessed using in silico analysis combined with RNA-Seq of miR-519e-mimic treated hiPSC-CMs and validated using Ago2-RNA immunoprecipitation(RIP) followed by qPCR. Results After transfection of miR-519e-mimics in hiPSC-­CM, a significant proliferative response indicative of cell division was observed in immunofluorescence staining and on protein level as compared with miR-scr treated hiPSC-CMs (EdU, H3P and AURKB, p&amp;lt;0.01). Apoptosis was blunted in miR-519e-treated hiPSC-CMs (p&amp;lt;0.05). MiR-519e-treatment of HAEC resulted in more meshes (p&amp;lt;0.01) and longer tube length (p&amp;lt;0.05), indicating enhanced pro-angiogenic capacity. Importantly, EdU-uptake and α-SMA were similar in miR-519e-treated cFB as compared to miR-scr, suggesting neutral effects of miR-519e on myofibroblast transition. In mice undergoing MI, injection of miR-519e-mimics significantly downregulated CDKN1A/P21(Cyclin dependent kinase inhibitor 1A) (p&amp;lt;0.05 vs. miR-scr), a direct target of miR-519e and negative master regulator of cell-cycling. For further novel target identification of miR-519e, we performed in silico analysis combined with RNA-Seq data from miR-519e-treated hiPSC-CM and validation using Ago2-RIP followed by qPCR. Herein, we identified PTEN(Phosphatase and tensin homolog) and TGFBR2(Transforming growth factor β receptor 2) that are involved in cardiomyocyte proliferation and hypoxic response, as direct targets of miR-519e. Conclusion Collectively, miR-519e is a potent inducer of cell division in human cardiomyocytes, with anti-apoptotic and pro-angiogenic capacity in the absence of pro-fibrotic effects. We show that miR-519e treatment downregulates key negative cell-cycle regulators in vivo and we identified novel direct targets of miR-519e that are involved in regulatory pathways for heart regeneration.

Long non-coding RNA PANDA promotes endothelial senescence and oxidative damage through the interaction with NRF2

Abstract Background Accumulation of reactive oxygen species and inflammation are major features of diabetic vasculopathy, yet the underlying mechanisms remain elusive. LncRNAs are emerging as important players in the pathogenesis of cardiovascular disease. PANDA, a newly identified lncRNA, is a key regulator of cellular senescence, apoptosis and oxidative stress. Purpose To investigate the role and molecular mechanism of PANDA in diabetic vascular disease. Methods RNAseq was performed to aorta specimen from diabetic and no diabetic patients as well to HAECs exposed to 5 mM and 25 mM concentrations (Normal -NG- and High -HG-, respectively). PANDA depletion in HG-treated HAECs was obtained by siRNA transfection while a scrambled siRNA wass used as a negative control. RNAseq and network perturbation analysis (NPA) of treated HAECs unveiled transcriptional changes upon PANDA depletion. Expression of PANDA was assessed by real time PCR. PANDA RNA-IP was performed to check its binding to relevant transcriptional factor (such as NRF2) as well Ch-IP was performed to check the NRF2 binding on oxidative gene promoters. Cellular localization of NRF2 was investigated by Immunofluorescence. Beta-galactosidase and superoxide staining was used to detect endothelial senescence and ROS formation; while, migration and tube formation were employed to evaluate angiogenic properties of HAECs. Results PANDA expression was significantly increased in diabetic human aorta as well in HG-treated HAECs (Figure1 A-B). Transcriptomic analysis revealed dysregulation of several genes upon PANDA silencing with the antioxidant gene heme oxygenase-1 (HMOX1) as the top-ranking transcript in HG cells (Figure1 C-D). Western blot analysis reveals the restored level of HMOX1 and TFRC1 upon PANDA depletion (Figure1 E-G). NPA analysis showed a strong involvement of PANDA in senescence, DNA damage, NRF2 signaling, hypoxic stress response and proliferation. In HG, NFR2 is downregulated while PANDA silencing restores its level (Figure1 I-J). We found that in HG condition PANDA binds the transcription factor NRF2 and blocks its nuclear translocation thus impeding its binding on HMOX1 and TFRC promoters (Figure1 K-M). Silencing of PANDA in HG-treated HAECs reduced genes and cellular features of senescence (Figure2 A-B), restored the expression of anti-apoptotic genes and decreases caspase 3 activity (Figure2 C-D), improved endothelial migration and tube formation (Figure2 E and G), and reduced ROS formation (Figure2 F). Conclusions In hyperglycemia PANDA upregulation drives endothelial senescence while impairing angiogenic properties. On the other hand, PANDA depletion in HG-treated HAECs rescues maladaptive transcriptional changes through the release of NRF2 that in turn translocate into the nucleus enhancing the expression of the antioxidant gene HMOX1. The results indicate PANDA as a putative novel molecular target in the setting of diabetic vascular disease (Figure2 H).

Glucose deprivation impairs hypoxia-inducible factor-1α synthesis

Hypoxia-inducible factors (HIFs) are key transcriptional mediators of the hypoxic response and are implicated in oncogenesis. HIFα is regulated by a well-characterized, oxygen-dependent degradation pathway involving the von Hippel Lindau (VHL) tumor suppressor protein. However, comparatively little is known about HIFα regulation at the translational level, particularly under cellular stress. There is evidence that HIFα expression not only responds to changes in oxygen tension, but also nutrient availability. In this study, we monitored global translation rates, ATP levels and HIF1α synthesis rates in response to glucose starvation or glycolysis inhibition. We found that both global and HIF1α-specific translation rates decline under glucose deprivation that is concomitant with ATP reduction. These results are in contrast with previous reports showing preferential HIF1α synthesis despite global translation suppression under hypoxia and suggest that a glucose requirement in cellular metabolism is associated with HIF1α translation.

Hypoxia reduces mouse urine output via HIF1α–mediated up–regulation of renal AQP1

Introduction: Patients with acute mountain sickness (AMS) due to hypoxia at high altitudes often exhibit abnormal water metabolism. Hypoxia-inducible factors (HIFs) are major regulators of adaptive responses to hypoxia. As transcription factors, HIFs are involved in the regulation of erythropoiesis, iron metabolism, angiogenesis, energy metabolism, and cell survival by promoting the transcriptional expression of hundreds of target genes. Roxadustat, a novel drug for the treatment of anemia associated with chronic kidney disease, acts by inhibiting the degradation of HIFs to increase their protein levels. However, the clinical use of roxadustat is frequently associated with peripheral edema, suggesting the involvement of HIFs in regulating the body's water balance possibly by modulating water reabsorption in the kidney. Methods: we first evaluated the effect of hypoxia (8% O2) on mouse urine output. We then performed in vitro experiments using hypoxia (1% O2) and roxadustat on mouse primary proximal tubular cells (mPTCs). The qPCR, western blot, and immunofluorescence were used to assess AQP1 mRNA and protein expression levels. Luciferase, CHIP, and EMSA were used to investigate the transcriptional regulation of AQP1 by HIF1α. Results: We found that mice exposed to hypoxia (8% O2) had significantly reduced urine volume compared to mice exposed to normoxia (21% O2). Hypoxia significantly elevated AQP1 expression at both mRNA and protein levels. In vitro experiments using mouse primary cultured proximal tubular cells (mPTCs) revealed that both hypoxia and roxadustat increased AQP1 expression. Mechanistically, overexpression of HIF1α, but not HIF2α, markedly increased AQP1 protein expression. Furthermore, the up-regulation of AQP1 by hypoxia and roxadustat can be blocked by the HIF1α inhibitor PX-478 in mPTCs. Finally, we found that the AQP1 gene promoter contains a putative hypoxia response element (HRE) and confirmed that AQP1 is a target gene of HIF1α using Luciferase reporter, CHIP, and EMSA assays. Conclusion: This study demonstrates that hypoxia can reduce the urine volume of mice via upregulating AQP1 expression by HIF1α in the proximal tubular epithelial cells. Our findings also suggest a potential mechanism involved in water metabolism disorders in patients with AMS and in patients with chronic kidney disease (CKD) receiving roxadustat treatment.

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells

AbstractCells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non‐heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2’s catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2’s gas tunnel. Using kinetic stopped‐flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5‐fold enhancement in its association rate to the iron center of tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced catalytic efficiency (kcat/KM=830±40 M−1 s−1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF‐1α, as compared to WT PHD2 (kcat/KM=90±9 M−1 s−1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK‐293T mammalian cells reveal significant reduction of HIF‐1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM=830±40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM=90±9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Higher oxygen content and transport characterize high-altitude ethnic Tibetan women with the highest lifetime reproductive success

We chose the "natural laboratory" provided by high-altitude native ethnic Tibetan women who had completed childbearing to examine the hypothesis that multiple oxygen delivery traits were associated with lifetime reproductive success and had genomic associations. Four hundred seventeen (417) women aged 46 to 86 y residing at ≥3,500 m in Upper Mustang, Nepal, provided information on reproductive histories, sociocultural factors, physiological measurements, and DNA samples for this observational cohort study. Simultaneously assessing multiple traits identified combinations associated with lifetime reproductive success measured as the number of livebirths. Women with the most livebirths had distinctive hematological and cardiovascular traits. A hemoglobin concentration near the sample mode and a high percent of oxygen saturation of hemoglobin raised arterial oxygen concentration without risking elevated blood viscosity. We propose ongoing stabilizing selection on hemoglobin concentration because extreme values predicted fewer livebirths and directional selection favoring higher oxygen saturation because higher values had more predicted livebirths. EPAS1, an oxygen homeostasis locus with strong signals of positive natural selection and a high frequency of variants occurring only among populations indigenous to the Tibetan Plateau, associated with hemoglobin concentration. High blood flow into the lungs, wide left ventricles, and low hypoxic heart rate responses aided effective convective oxygen transport to tissues. Women with physiologies closer to unstressed, low altitude values had the highest lifetime reproductive success. This example of ethnic Tibetan women residing at high altitudes in Nepal links reproductive fitness with trait combinations increasing oxygen delivery under severe hypoxic stress and demonstrates ongoing natural selection.