Pathophysiological Hypoxia Research Articles

Hypoxia/HIF Modulates Immune Responses.

Oxygen availability varies throughout the human body in health and disease. Under physiological conditions, oxygen availability drops from the lungs over the blood stream towards the different tissues into the cells and the mitochondrial cavities leading to physiological low oxygen conditions or physiological hypoxia in all organs including primary lymphoid organs. Moreover, immune cells travel throughout the body searching for damaged cells and foreign antigens facing a variety of oxygen levels. Consequently, physiological hypoxia impacts immune cell function finally controlling innate and adaptive immune response mainly by transcriptional regulation via hypoxia-inducible factors (HIFs). Under pathophysiological conditions such as found in inflammation, injury, infection, ischemia and cancer, severe hypoxia can alter immune cells leading to dysfunctional immune response finally leading to tissue damage, cancer progression and autoimmunity. Here we summarize the effects of physiological and pathophysiological hypoxia on innate and adaptive immune activity, we provide an overview on the control of immune response by cellular hypoxia-induced pathways with focus on the role of HIFs and discuss the opportunity to target hypoxia-sensitive pathways for the treatment of cancer and autoimmunity.

Abstract 2650: Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells

Abstract BACKGROUND: Glioblastoma (GBM) is the most common and malignant form of primary brain tumor in adults and remains incurable. Inter-organelle communication between mitochondria and nuclei is a potential compensatory signaling pathway that may contribute to therapeutic resistance and cellular plasticity in the face of a hypoxic tumor microenvironment. Coiled-coil-helix-coiled-coil-helix domain-containing protein 2 (CHCHD2) is a protein that colocalizes to the mitochondrial inter-membrane space and the nucleus, where it exhibits hypoxia-sensitive transcription factor capabilities. We demonstrated previously that CHCHD2 knockout in hypoxic U87 GBM cells harboring epidermal growth factor receptor (EGFR) variant III (U87vIII) abrogated the increase in proliferation and invasion exhibited by U87vIII WT cells expressing CHCHD2. The C-terminus of CHCHD2 contains redox-sensitive cysteines amenable to thiol/disulfide formation, which determine protein folding. The objective of this study was to characterize the effect of hypoxia and cellular redox status on CHCHD2 subcellular localization. We hypothesized that reducing the C-terminal CX9C motif would induce CHCHD2 mitochondrial export and nuclear accumulation. METHODS: U87 and U87vIII cells were incubated in standard culture oxygen conditions (~21% O2), 7% O2, 4% O2, and pathophysiological hypoxia (1% O2). Confocal immunofluorescence microscopy was used to observe CHCHD2 subcellular localization, and nuclear fluorescence intensity was quantified using Axiovision software. Western blot of whole cell lysates was used to quantify CHCHD2 protein levels in response to decreasing oxygen tensions. Chemical reduction of cells was achieved using dithiothreitol (DTT) at designated concentrations and time courses. RESULTS: The amount of nuclear CHCHD2 did not differ between 21% and 7% O2. Incubation in 1% O2 increased the amount of nuclear CHCHD2 in both U87 and U87vIII cells, with the latter consistently exhibiting greater amounts of nuclear CHCHD2 at all oxygen tensions. Total CHCHD2 protein levels in U87 cells were maximal at 4% O2, but reduced at 1% O2, a trend also observed in U87vIII cells. Chemical reduction with 1 mM DTT for either 15 min or 24 h induced significantly greater CHCHD2 nuclear accumulation in U87vIII cells compared to U87. Increasing DTT dose 10-fold resulted in rapid accumulation of CHCHD2 in nuclei after 15 min, and complete disappearance from mitochondria after 30 min. CONCLUSIONS: Hypoxia and chemical reduction are capable of driving CHCHD2 translocation from mitochondria to the nucleus in U87 GBM cells, which is enhanced in the presence of the EGFRvIII mutant. Ongoing work to expand our understanding of how CHCHD2 protein folding governs protein localization and function may inspire novel therapeutic strategies to undermine mitonuclear signaling in GBM. Citation Format: Jan C. Lumibao, H. Rex Gaskins. Characterizing CHCHD2 subcellular localization in response to hypoxia and redox perturbations in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2650.

Hypoxia induces production of citrullinated proteins in human fibroblast-like synoviocytes through regulating HIF1α.

Hypoxia is a prominent microenvironment feature in a range of disorders including cancer, rheumatoid arthritis (RA), atherosclerosis, inflammatory bowel disease (IBD), infection and obesity. Hypoxia promotes biological functions of fibroblast-like synoviocytes via regulating hypoxia-inducible factor 1α (HIF1α). Dysregulated protein citrullination in RA drives the production of antibodies to citrullinated proteins, a highly specific biomarker of RA. However, the mechanisms promoting citrullination in RA are not yetfullyelucidated. In this study, we investigated whether pathophysiological hypoxia as found in the rheumatoid synovium modulates the citrullination in human fibroblast-like synoviocytes (HFLS). Here, we found that peptidylarginine deiminase 2 (PAD2) and citrullinated proteins were increased in HFLS after exposure to hypoxia. Moreover, knocking down HIF1α by HIF1α siRNA ameliorated the expression of PAD2 and citrullinated proteins. Collectively, this study provides a new mechanism involved in generating citrullinated proteins: hypoxia promotes citrullination and PAD production in HFLS. Concurrently, we also proposed a novel hypoxia involved mechanism in RA pathogenesis. This study deepens our understanding of the role of hypoxia in the pathogenesis of RA and provides a potential therapeutic strategy for RA.

Oxygen concentration modulates cellular senescence and autophagy in human trophoblast cells.

We investigated the effect of oxygen concentrations on cellular senescence and autophagy and examined the role of autophagy in human trophoblast cells. Human first-trimester trophoblast cells (Sw.71) were incubated under 21%, 5%, or 1% O2 concentrations for 24hours. We examined the extent of senescence caused using senescence-associated β-galactosidase (SA-β-Gal) and senescence-associated secretory phenotype (SASP) as markers. Moreover, we examined the role of autophagy in causing cellular senescence using an autophagy inhibitor (3-methyladenine, 3MA). Physiological normoxia (5% O2 ) decreased SA-β-Gal-positive cells and SASP including interleukin-6 (IL-6) and IL-8 compared with cultured cells in 21% O2 . Pathophysiological hypoxia (1% O2 ) caused cytotoxicity, including extracellular release of ATP and lactate dehydrogenase, and decreased senescence phenotypes. 3MA-treated trophoblast cells significantly suppressed senescence markers (SA-β-Gal-positive cells and SASP secretion) in O2 -independent manner. We conclude that O2 concentration modulates cellular senescence phenotypes regulating autophagy in the human trophoblast cells. Moreover, inhibiting autophagy suppresses cellular senescence, suggesting that autophagy contributes to oxygen stress-induced cellular senescence.

A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

Intracellular O2 is a key regulator of NO signaling, yet most in vitro studies are conducted in atmospheric O2 levels, hyperoxic with respect to the physiologic milieu. We investigated NO signaling in endothelial cells cultured in physiologic (5%) O2 and stimulated with histamine or shear stress. Culture of cells in 5% O2 (>5 d) decreased histamine- but not shear stress–stimulated endothelial (e)NOS activity. Unlike cells adapted to a hypoxic environment (1% O2), those cultured in 5% O2 still mobilized sufficient Ca2+ to activate AMPK. Enhanced expression and membrane targeting of PP2A-C was observed in 5% O2, resulting in greater interaction with eNOS in response to histamine. Moreover, increased dephosphorylation of eNOS in 5% O2 was Ca2+-sensitive and reversed by okadaic acid or PP2A-C siRNA. The present findings establish that Ca2+ mobilization stimulates both NO synthesis and PP2A-mediated eNOS dephosphorylation, thus constituting a novel negative feedback mechanism regulating eNOS activity not present in response to shear stress. This, coupled with enhanced NO bioavailability, underpins differences in NO signaling induced by inflammatory and physiologic stimuli that are apparent only in physiologic O2 levels. Furthermore, an explicit delineation between physiologic normoxia and genuine hypoxia is defined here, with implications for our understanding of pathophysiological hypoxia.—Keeley, T. P., Siow, R. C. M., Jacob, R., Mann, G. E. A PP2A-mediated feedback mechanism controls Ca2+-dependent NO synthesis under physiological oxygen.

Hypoxia and cellular metabolism in tumour pathophysiology.

Cancer cells are optimised for growth and survival via an ability to outcompete normal cells in their microenvironment. Many of these advantageous cellular adaptations are promoted by the pathophysiological hypoxia that arises in solid tumours due to incomplete vascularisation. Tumour cells are thus faced with the challenge of an increased need for nutrients to support the drive for proliferation in the face of a diminished extracellular supply. Among the many modifications occurring in tumour cells, hypoxia inducible factors (HIFs) act as essential drivers of key pro-survival pathways via the promotion of numerous membrane and cytosolic proteins. Here we focus our attention on two areas: the role of amino acid uptake and the handling of metabolic acid (CO2 /H+ ) production. We provide evidence for a number of hypoxia-induced proteins that promote cellular anabolism and regulation of metabolic acid-base levels in tumour cells including amino-acid transporters (LAT1), monocarboxylate transporters, and acid-base regulating carbonic anhydrases (CAs) and bicarbonate transporters (NBCs). Emphasis is placed on current work manipulating multiple CA isoforms and NBCs, which is at an interesting crossroads of gas physiology as they are regulated by hypoxia to contribute to the cellular handling of CO2 and pHi regulation. Our research combined with others indicates that targeting of HIF-regulated membrane proteins in tumour cells will provide promising future anti-cancer therapeutic approaches and we suggest strategies that could be potentially used to enhance these tactics.

Cell cycle progression in glioblastoma cells is unaffected by pathophysiological levels of hypoxia.

Hypoxia is associated with the increased malignancy of a broad range of solid tumours. While very severe hypoxia has been widely shown to induce cell cycle arrest, the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this study was to investigate the effect of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an extremely aggressive brain tumour with a heterogeneous oxygenation pattern. The effects of a range of oxygen tensions on GBM cell lines and primary cells were assessed using flow cytometry. Results indicate that cell cycle distribution and viability are unaffected by long term exposure (24–96 h) to pathophysiological levels of oxygen (1–8% O2). Both transient cell cycle arrest and small amounts of cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). No significant changes in p21 protein expression levels were detected. These findings reinforce the importance of using physiologically relevant oxygen tensions when investigating tumour hypoxia, and help to explain how solid tumours can be both hypoxic and highly proliferative, as is the case with GBM.

γ₁34.5-deleted HSV-1-expressing human cytomegalovirus IRS1 gene kills human glioblastoma cells as efficiently as wild-type HSV-1 in normoxia or hypoxia.

Pathophysiological hypoxia, which fosters the glioma stem-like cell (GSC) phenotype, is present in high-grade gliomas and has been linked to tumor development, invasiveness, and resistance to chemotherapy and radiation. Oncolytic virotherapy with engineered herpes simplex virus-1 (HSV-1) is a promising therapy for glioblastoma; however efficacy of γ134.5-deleted HSVs, which have been used in clinical trials, was diminished in hypoxia. We investigated the ability of a chimeric HCMV/HSV-1 virus, which expresses the human CMV PKR evasion gene IRS1 and is in preparation for clinical trials, to infect and kill adult and pediatric patient-derived glioblastoma xenografts in hypoxia and normoxia. Infectivity, cytotoxicity and viral recovery were significantly greater with the chimeric virus compared to the γ134.5-deleted virus, regardless of oxygen tension. The chimeric virus infected and killed CD133+ GSCs similarly to wild-type HSV-1. Increased activation of mitogen-activated protein kinase (MAPK) p38 and its substrate heat shock protein 27 (Hsp27) was seen after viral infection in normoxia compared to hypoxia. Hsp27 knockdown or p38 inhibition reduced virus recovery indicating that the p38 pathway plays a role in the reduced efficacy of the γ134.5-deleted virus in hypoxia. Together these findings demonstrate chimeric HCMV/HSV-1 efficiently targets both CD133+ GSCs and glioma cells in hypoxia.

P58 * BRAIN TUMOUR CELLS CAN SURVIVE AND THRIVE IN THE HYPOXIC SOLID TUMOUR MICROENVIRONMENT

INTRODUCTION: Normal brain tissue is exposed to 5-8% oxygen whilst the average oxygen level within brain tumours is just 1% (hypoxia). Hypoxia is associated with the increased malignancy, invasiveness, and treatment resistance of brain tumours. Paradoxically, hypoxia has also been shown to induce cell cycle arrest in certain cells; however the impact of pathophysiological hypoxia on tumour cell proliferation is poorly understood. The aim of this project was to investigate the consequences of different oxygen levels on glioblastoma (GBM) cell proliferation and survival. GBM is an aggressive brain tumour with an extremely poor prognosis. METHOD: GBM cell lines were exposed to a range of oxygen tensions and their cell cycle distribution and survival was assessed using flow cytometry. Changes in the expression level of genes involved in regulation of the cell cycle were quantified by qRT-PCR. RESULTS: Results indicate that GBM proliferation and survival is unaffected by long-term exposure to pathophysiological levels of oxygen (1% O2). Cell cycle arrest and/or cell death could only be detected when cells were exposed to severe hypoxia (0.1% O2). Sensitivity to severe hypoxia was associated with increased expression of the cyclin-dependent kinase inhibitor protein p21. CONCLUSION: These findings demonstrate that the pro-tumorigenic effects of pathophysiological hypoxia are not balanced out by reduced cellular proliferation, strengthening the association between hypoxia and tumour malignancy. This helps to explain how a hypoxic tumour such as GBM can be incredibly fast growing and aggressive.

AB0026 Pathophysiological hypoxia affects both redox state and il-2 signaling of human cd4+ t cells and concomitantly impairs survival and proliferation

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease of diarthrodial joints, characterized by the infiltration of immune cells, alterations of cellular redox state and pathophysiological local tissue hypoxia (<1% O2).ObjectivesWe...

Pathophysiological hypoxia affects the redox state and IL‐2 signalling of human CD4+ T cells and concomitantly impairs survival and proliferation

Inflamed areas are characterized by infiltration of immune cells, local hypoxia and alterations of cellular redox states. We investigated the impact of hypoxia on survival, proliferation, cytokine secretion, intracellular energy and redox state of human CD4(+) T cells. We found that pathophysiological hypoxia (<2% O2 ) significantly decreased CD4(+) T-cell survival after mitogenic stimulation. This effect was not due to an increased caspase-3/7-mediated apoptosis or adenosine-5'-triphosphate (ATP) consumption/depletion. However, the ability of stimulated T cells to proliferate was reduced under hypoxic conditions, despite increased expression of CD25. Pathophysiological hypoxia was also found to modify intracellular ROS (iROS) levels in stimulated T cells over time as compared with levels found in normoxia. Physiological hypoxia (5% O2 ) did not decrease CD4(+) T-cell survival and proliferation or modify iROS levels as compared with normoxia. We conclude that pathophysiological hypoxia affects T-cell proliferation and viability via disturbed IL-2R signalling downstream of STAT5a phosphorylation, but not as a result of impaired cellular energy homeostasis. We suggest iROS links early events in T-cell stimulation to the inhibition of the lymphoproliferative response under pathophysiological hypoxic conditions. The level of iROS may therefore act as a mediator of immune functions leading to down-regulation of long-term T-cell activity in inflamed tissues.

Effects of developmental hypoxia on alligator cardiac myocytes

Pathophysiological hypoxia induces changes in the structure, function, and gene expression of the developing heart. Recent work in reptiles exposed to hypoxia during development revealed cardiac hypertrophy and a reduction in both resting heart rate and blood pressure. The purpose of the current study was to further investigate these responses at the level of the cardiac myocyte. America alligators (Alligator mississippiensis) were reared in normoxic and hypoxic conditions until 90% development. The heart was quickly excised and single ventricular myocytes were liberated via a modified langendorf perfusion using digestive enzymes. Ventricular myocytes were long and thin with clear sarcomeric striations. Sarcomeric shortening and calcium transients were simultaneously measured as contraction frequency was increased from 0.5 to 2.0 Hz. Data are presented as means ± SEM from n=7–15 cells under each condition from N=20 animals. There were no differences between normoxic and hypoxic resting sarcomere length (SL) (1.78±0.04 and 1.77±0.05 um, respectively) and resting SL decreased (~3%) in both treatments with increased frequency. SL shortening was significantly greater in the hypoxic (9.9±1.4%) than the normoxic (4.6±1.4%) myocytes at 0.5 Hz but the differences were reduced as frequency increased. Calcium transient amplitude data mirrored that of sarcomeric shortening.

Adaptation of Human CD4+ T Cells to Pathophysiological Hypoxia: A Transcriptome Analysis

Inflamed tissues are usually characterized by low oxygen levels. We investigated whether pathophysiological hypoxia (pO(2) < 1%) as found in the rheumatoid synovium modulates the transcriptome of human CD4+ T cells. We analyzed the extent to which hypoxia influences the transcriptome in the rheumatoid synovium according to a gene cluster reflecting adaptation to low oxygen levels. Hypoxia-inducible factor-1alpha (HIF-1alpha) was detected in the rheumatoid synovium using immunohistochemistry. Isolated human CD4+ T cells were exposed to hypoxia and analyzed using microarray analysis, quantitative polymerase chain reaction, and immunoblot detection. In rheumatoid arthritis (RA) synovial tissue samples, hypoxia modulates the transcription profile. This profile is similar, but not identical, to that found in isolated CD4+ T cells incubated under hypoxic conditions. We show that HIF-1alpha is expressed in synovial tissue samples and in hypoxic CD4+ cells; and that hypoxia directly affects differential gene expression in human T cells with up to 4.8% modulation of the transcriptome. Functional genome analysis revealed substantial effects of hypoxia on immune response, transcriptional regulation, protein modification, cell growth and proliferation, and cell metabolism. Severe hypoxia, a feature of joint inflammation, considerably modulates the transcriptome of cells found in the rheumatoid synovium. Human CD4+ T cells adapt to hypoxic conditions mainly by HIF-1-driven effects on the transcriptome reflecting a profound influence on immune functions. Thus, hypoxia must be taken into account when therapeutically targeting inflammation.

Mitogen-activated Protein Kinase Phosphatase-1 (MKP-1) Expression Is Induced by Low Oxygen Conditions Found in Solid Tumor Microenvironments: A CANDIDATE MKP FOR THE INACTIVATION OF HYPOXIA-INDUCIBLE STRESS-ACTIVATED PROTEIN KINASE/c-Jun N-TERMINAL PROTEIN KINASE ACTIVITY

Pathophysiological hypoxia is an important modulator of gene expression in solid tumors and other pathologic conditions. We observed that transcriptional activation of the c-jun proto-oncogene in hypoxic tumor cells correlates with phosphorylation of the ATF2 transcription factor. This finding suggested that hypoxic signals transmitted to c-jun involve protein kinases that target AP-1 complexes (c-Jun and ATF2) that bind to its promoter region. Stress-inducible protein kinases capable of activating c-jun expression include stress-activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK) and p38 members of the mitogen-activated protein kinase (MAPK) superfamily of signaling molecules. To investigate the potential role of MAPKs in the regulation of c-jun by tumor hypoxia, we focused on the activation SAPK/JNKs in SiHa human squamous carcinoma cells. Here, we describe the transient activation of SAPK/JNKs by tumor-like hypoxia, and the concurrent transcriptional activation of MKP-1, a stress-inducible member of the MAPK phosphatase (MKP) family of dual specificity protein-tyrosine phosphatases. MKP-1 antagonizes SAPK/JNK activation in response to diverse environmental stresses. Together, these findings identify MKP-1 as a hypoxia-responsive gene and suggest a critical role in the regulation of SAPK/JNK activity in the tumor microenvironment.

The redox-sensitive human antioxidant responsive element induces gene expression under low oxygen conditions.

Transient transfection studies of human HepG2 and mouse Hepa hepatocarcinoma cells with a reporter gene construct regulated by a human antioxidant responsive element (ARE) from the NQO1 gene demonstrated that the element is responsive to low oxygen conditions. The antioxidant N-acetyl L-cysteine (NAC) strongly inhibited basal aerobic reporter gene activity in HepG2 cells without obviously affecting the hypoxic induction, as is consistent with ARE sensitivity to oxidative stress in aerobic cultures. Electrophoretic mobility shift (EMS) assays of nuclear extracts of HepG2 and Hepa cells lysed under aerobic or hypoxic conditions or after exposure to the phenolic compound 3-(2)-tert-butyl-4-hydroxyanisole (BHA), showed specific and constitutive protein binding to the ARE under all of these conditions. Taken together, these findings show that the ARE can mediate gene expression in response to low oxygen conditions. Co-ordinately regulated expression of ARE-dependent genes, such as phase II detoxification enzymes, may be an important phenotype of solid tumors containing significant regions of pathophysiological hypoxia.