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1.  Hyaluronic acid hydrogel stiffness and oxygen tension affect cancer cell fate and endothelial sprouting 
Biomaterials science  2014;2(5):655-665.
Three-dimensional (3D) tissue culture models may recapitulate aspects of the tumorigenic microenvironment in vivo, enabling the study of cancer progression in vitro. Both hypoxia and matrix stiffness are known to regulate tumor growth. Using a modular culture system employing an acrylated hyaluronic acid (AHA) hydrogel, three hydrogel matrices with distinctive degrees of viscoelasticity — soft (78±16 Pa), medium (309± 57 Pa), and stiff (596± 73 Pa) — were generated using the same concentration of adhesion ligands. Oxygen levels within the hydrogel in atmospheric (21 %), hypoxic (5 %), and severely hypoxic (1 %) conditions were assessed with a mathematical model. HT1080 fibrosarcoma cells, encapsulated within the AHA hydrogels in high densities, generated nonuniform oxygen distributions, while lower cell densities resulted in more uniform oxygen distributions in the atmospheric and hypoxic environments. When we examined how varying viscoelasticity in atmospheric and hypoxic environments affects cell cycles and the expression of BNIP3 and BNIP3L (autophagy and apoptosis genes), and GLUT-1 (a glucose transport gene), we observed that HT1080 cells in 3D hydrogel adapted better to hypoxic conditions than those in a Petri dish, with no obvious correlation to matrix viscoelasticity, by recovering rapidly from possible autophagy/apoptotic events and alternating metabolism mechanisms. Further, we examined how HT1080 cells cultured in varying viscoelasticity and oxygen tension conditions affected endothelial sprouting and invasion. We observed that increased matrix stiffness reduced endothelial sprouting and invasion in atmospheric conditions; however, we observed increased endothelial sprouting and invasion under hypoxia at all levels of matrix stiffness with the upregulation of vascular endothelial growth factor (VEGF) and angiopoeitin-1 (ANG-1). Overall, HT1080 cells encapsulated in the AHA hydrogels under hypoxic stress recovered better from apoptosis and demonstrated greater angiogenic induction. Thus, we propose that oxygen tension more profoundly influences cell fate and the angiogenic potential of 3D cultured HT1080 fibrosarcoma cells than does matrix stiffness.
PMCID: PMC3987918  PMID: 24748963
2.  Hypoxic Preconditioning Results in Increased Motility and Improved Therapeutic Potential of Human Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2008;26(8):2173-2182.
Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential.
PMCID: PMC3017477  PMID: 18511601
Immune-deficient mice; Human stem cells; Mesenchymal stem cells; Hypoxia; Transplantation; Tissue repair
3.  Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. 
Journal of Clinical Investigation  1997;99(7):1729-1738.
Clinical conditions associated with local or systemic hypoxemia can lead to prothrombotic diatheses. This study was undertaken to establish a model of whole-animal hypoxia wherein oxygen deprivation by itself would be sufficient to trigger tissue thrombosis. Furthermore, this model was used to test the hypothesis that hypoxia-induced mononuclear phagocyte (MP) recruitment and tissue factor (TF) expression may trigger the local deposition of fibrin which occurs in response to oxygen deprivation. Using an environmental chamber in which inhaled oxygen tension was lowered to 6%, hypoxic induction of thrombosis was demonstrated in murine pulmonary vasculature by 8 h based upon: (a) immunohistologic evidence of fibrin formation in hypoxic lung tissue using an antifibrin antibody, confirmed by 22.5-nm strand periodicity by electron microscopy; (b) immunoblots revealing fibrin gamma-gamma chain dimers in lungs from hypoxic but not normoxic mice or hypoxic mice treated with hirudin; (c) accelerated deposition of 125I-fibrin/fibrinogen and 111In-labeled platelets in the lung tissue of hypoxic compared with normoxic animals; (d) reduction of tissue 125I-fibrin/fibrinogen accumulation in animals which had either been treated with hirudin or depleted of platelets before hypoxic exposure. Because immunohistochemical analysis of hypoxic pulmonary tissue revealed strong MP staining for TF, confirmed by increased TF RNA in hypoxic lungs, and because 111In-labeled murine MPs accumulated in hypoxic pulmonary tissue, we evaluated whether recruited MPs might be responsible for initiation of hypoxia-induced thrombosis. This hypothesis was supported by several lines of evidence: (a) MP depletion before hypoxia reduced thrombosis, as measured by reduced 125I-fibrin/fibrinogen deposition and reduced accumulation of cross-linked fibrin by immunoblot; (b) isolated murine MPs demonstrated increased TF immunostaining when exposed to hypoxia; and (c) administration of an anti-rabbit TF antibody that cross-reacts with murine TF decreased 125I-fibrin/fibrinogen accumulation and cross-linked fibrin accumulation in response to hypoxia in vivo. In summary, these studies using a novel in vivo model suggest that MP accumulation and TF expression may promote hypoxia-induced thrombosis.
PMCID: PMC507994  PMID: 9120018
4.  Tuberculous Granulomas Are Hypoxic in Guinea Pigs, Rabbits, and Nonhuman Primates▿  
Infection and Immunity  2008;76(6):2333-2340.
Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O2. Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 ± 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.
PMCID: PMC2423064  PMID: 18347040
5.  Increased Proliferation and Analysis of Differential Gene Expression in Human Wharton's Jelly-derived Mesenchymal Stromal Cells under Hypoxia 
Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.
PMCID: PMC2945278  PMID: 20877435
Hypoxia; Wharton's jelly; Mesenchymal stem cells (MSCs); Transcription; Transformation markers; Cell proliferation.
6.  Hypoxic Stress Induced TREM-1 and Inflammatory Chemokines in Human Peripheral Blood Mononuclear Cells 
Hypoxia is a condition of low pO2, which creates a unique microenvironment affecting cell phenotype and subsequent immune response generation. Little is known about the impact of hypoxia on the phenotypic expression of NK cell, TREM-1, TLR-4 and inflammatory chemokines. In the present study we have determined the frequency of peripheral blood populations of CD16/CD56 (NK Cells) expressing cells, presence of activation marker CD354 (TREM-1), Toll like receptor (CD 284) on the cell surface and chemokines IL-8 and RANTES in the cellular supernatant of normoxia and hypoxia exposed cells by flow cytometry. GRP-78 expression was determined by reverse transcriptase polymerase chain reaction. The blood was collected from healthy individuals and exposed to normoxic and hypoxic (0.5 %) environment for 24 h. The percentage of NK cells (CD 16/56) was marginally up regulated while TLR-4 expression was diminished in hypoxia exposed cells as compare to the normoxic cells. TREM-1 expression was significantly up-regulated (p < 0.05) in hypoxia as compared to the normoxic control. In addition when monocytic cell line THP-1 was exposed to 0.5 % hypoxia for 24 h, TLR4 expression was significantly decreased in hypoxic cells as compared to normoxic cells. Furthermore, GRP-78 mRNA expression was also upregulated by hypoxia or LPS exposure. These events are paralleled by strengthening up-regulation of the chemokines IL-8 and RANTES an otherwise necessary event for the chemotaxis of the neutrophils and macrophages to the inflammatory site. In conclusion, this study provides a novel insight into the mechanism linking low oxygen tension to the regulation of immune and inflammatory responses, leading to new perspectives of the role of hypoxia in programming immune cell functions.
PMCID: PMC3990790  PMID: 24757292
Hypoxia; GRP-78; Chemokine; IL-8; RANTES; Inflammation
7.  Reoxygenation of hypoxia-differentiated dentritic cells induces Th1 and Th17 cell differentiation 
Molecular immunology  2009;47(4):922.
Dendritic cells (DCs) are often exposed to various oxygen tensions under physiological and pathological conditions. However, the effects of various oxygen tensions on DC functions remain unclear. In this study, we showed that hypoxia-differentiated DCs expressed lower levels of MHC-II molecule, co-stimulatory molecules (CD80, CD86) and proinflammatory cytokines (IL-1β, IL-6, and TNF-α), but higher levels of immunoregulatory cytokine transforming growth factor-beta (TGF-β) than normoxia-differentiated DCs. Unexpectedly, re-exposure of hypoxia-differentiated DCs to saturated oxygen (reoxygenation) completely restored their mature phenotype and function. Specifically, the reoxygenated DCs induced naïve CD4+ T cells to differentiate into Th1 and Th17 effector cells, but deceased the generation of CD4+CD25+Foxp3+ regulatory T cells (Tregs). The data indicate that hypoxic microenvironment suppresses the maturation and function of murine DCs. Reoxygenation of hypoxia-differentiated DCs however results in complete recovery of their mature phenotype and function, and has strong ability to drive immune response toward a proinflammatory direction, suggesting reoxygenated DCs may contribute to inflammation of ischemia-reperfusion injury.
PMCID: PMC2815172  PMID: 19910049
Dendritic cells; Inflammation; Foxp3+ Tregs; IL-6; TGF-β; Hypoxia
8.  Direct measurement of local oxygen concentration in the bone marrow of live animals 
Nature  2014;508(7495):269-273.
Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.
PMCID: PMC3984353  PMID: 24590072
9.  In vivo Hypoxia and a Fungal Alcohol Dehydrogenase Influence the Pathogenesis of Invasive Pulmonary Aspergillosis 
PLoS Pathogens  2011;7(7):e1002145.
Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and 1H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.
Author Summary
Metabolic flexibility is important for human pathogens like Aspergillus fumigatus as it allows adaptation to dynamic infection induced microenvironments. Consequently, identification of fungal metabolic pathways critical for in vivo growth may uncover novel virulence mechanisms and new therapeutic opportunities. To date, the mechanisms used by A. fumigatus to adapt to microenvironments in immunosuppressed mammalian hosts are poorly understood. In this study we discover that A. fumigatus is exposed to oxygen limiting microenvironments during invasive pulmonary aspergillosis (IPA). Thus, this result builds on growing evidence that suggests hypoxia is a significant in vivo stress encountered by human fungal pathogens. We tested the hypothesis that genes encoding enzymes involved in ethanol fermentation are important for in vivo fungal responses to hypoxia. We consequently observed a significant increase in the inflammatory response that correlated with reduced fungal growth in the lungs of mice inoculated with an alcohol dehydrogenase null mutant. Altogether, our study suggests that fungal responses to in vivo hypoxic microenvironments can directly affect host immune responses to the invading fungal pathogen. A better understanding of these mechanisms will increase our understanding of IPA and other human diseases caused by fungi and could potentially lead to improved therapeutic options.
PMCID: PMC3141044  PMID: 21811407
10.  Coordinated induction of plasminogen activator inhibitor-1 (PAI-1) and inhibition of plasminogen activator gene expression by hypoxia promotes pulmonary vascular fibrin deposition. 
Journal of Clinical Investigation  1998;102(5):919-928.
Oxygen deprivation, as occurs during tissue ischemia, tips the natural anticoagulant/procoagulant balance of the endovascular wall to favor activation of coagulation. To investigate the effects of low ambient oxygen tension on the fibrinolytic system, mice were placed in a hypoxic environment with pO2 < 40 Torr. Plasma levels of plasminogen activator inhibitor-1 (PAI-1) antigen, detected by ELISA, increased in a time-dependent fashion after hypoxic exposure (increased as early as 4 h, P < 0.05 vs. normoxic controls), and were accompanied by an increase in plasma PAI-1 activity by 4 h (P < 0.05 vs. normoxic controls). Northern analysis of hypoxic murine lung demonstrated an increase in PAI-1 mRNA compared with normoxic controls; in contrast, transcripts for both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) decreased under hypoxic conditions. Immunocolocalization studies identified macrophages as the predominant source of increased PAI-1 within hypoxic lung. Using a transformed murine macrophage line, striking induction of PAI-1 transcripts occurred under hypoxic conditions, due to both increased de novo transcription as well as increased mRNA stability. Consistent with an important role of the fibrinolytic system in hypoxia-induced fibrin accumulation, PAI-1 +/+ mice exposed to hypoxia exhibited increased pulmonary fibrin deposition based upon a fibrin immunoblot, intravascular fibrin identified by immunostaining, and increased accumulation of 125I-fibrinogen/fibrin in hypoxic tissue. In contrast, mice deficient for the PAI-1 gene (PAI-1 -/-) similarly exposed to hypoxic conditions did not display increased fibrin accumulation compared with normoxic PAI-1 +/+ controls. Furthermore, homozygous null uPA (uPA -/-) and tPA (tPA -/-) mice subjected to oxygen deprivation showed increased fibrin deposition compared with wild-type controls. These studies identify enhanced expression of PAI-1 as an important mechanism suppressing fibrinolysis under conditions of low oxygen tension, a response which may be further amplified by decreased expression of plasminogen activators. Taken together, these data provide insight into an important potential role of macrophages and the fibrinolytic system in ischemia-induced thrombosis.
PMCID: PMC508957  PMID: 9727060
11.  Oxygen Tension Variation in Ischemic Gastrocnemius Muscle, Marrow, and different Hypoxic Conditions In Vitro 
Bone marrow stromal cells (BMSCs) play an important role in ischemic limb angiogenesis. BMSCs cultured in vitro can be exposed to oxygen tension much higher than that experienced in vivo. This study assessed oxygen tension in bone marrow and ischemic muscle in vivo, and then identified an appropriate oxygen concentration for culturing BMSCs.
Unilateral hind limb ischemia was surgically induced in 30 mice, and tissue oxygen tension in bilateral gastrocnemius muscles and femoral bone marrow was monitored in vivo using a micro-electrode at 24 hours, 1 week, 2 weeks, and 3 weeks after modeling. Media used for culturing normal marrow, muscle, and artery tissue were incubated with various oxygen concentrations, and O2 tension was continuously monitored. Oxygen tension in aortic arterial blood was monitored using a micro-electrode and blood gas analyzer, and the results were compared.
Oxygen tension in ischemic gastrocnemius muscle reached a nadir at 1 week after ischemic modeling, when histological changes were most noticeable. Culture media incubated with 3%, 6%, and 14% oxygen (the normal oxygen levels of bone marrow, muscle, and arterial blood, respectively) required 9, 6, and 2 hours, respectively, to reach an equilibrated oxygen tension, and oxygen tension was elevated by 1.6-, 1.2-, and 0.4-fold, respectively, upon re-exposure of the media to air.
Physiological oxygen tension differs in different tissues. A 3% O2 concentration mimics the physiological O2 exposure experienced by BMSCs and represents the hypoxic concentration. Culture medium incubated under hypoxic conditions requires a prolonged period of time to regain equilibrated oxygen tension.
PMCID: PMC4301230  PMID: 25372971
Arterial Pressure; Cell Hypoxia; Mesenchymal Stromal Cells
12.  NADPH Oxidase 2 Regulates Bone Marrow Microenvironment Following Hindlimb Ischemia: Role in Reparative Mobilization of Progenitor Cells 
Stem cells (Dayton, Ohio)  2012;30(5):923-934.
Bone marrow (BM) microenvironment, which is regulated by hypoxia and proteolytic enzymes, is crucial for stem/progenitor cell function and mobilization involved in postnatal neovascularization. We demonstrated that NADPH oxidase2 (Nox2)-derived reactive oxygen species (ROS) are involved in post-ischemic mobilization of BM cells and revascularization. However, role of Nox2 in regulating BM microenvironment in response to ischemic injury remains unknown. Here we show that hindlimb ischemia of mice increases ROS production in both the endosteal and central region of BM tissue in situ, which is almost completely abolished in Nox2 knockout (KO) mice. This Nox2-dependent ROS production is mainly derived from Gr-1+ myeloid cells in BM. In vivo injection of hypoxyprobe reveals that endosteum at the BM is hypoxic with high expression of HIF-1α in basal state. Following hindlimb ischemia, hypoxic areas and HIF-1α expression are expanded throughout the BM, which is inhibited in Nox2 KO mice. This ischemia-induced alteration of Nox2-dependent BM microenvironment is associated with an increase in VEGF expression and Akt phosphorylation in BM tissue, thereby promoting Lin− progenitor cell survival and expansion, leading to their mobilization from BM. Furthermore, hindlimb ischemia increases proteolytic enzymes MT1-MMP expression and MMP-9 activity in BM, which is inhibited in Nox2 KO mice. In summary, Nox2-dependent increase in ROS play a critical role in regulating hypoxia expansion and proteolytic activities in BM microenvironment in response to tissue ischemia. This in turn promotes progenitor cell expansion and reparative mobilization from BM, leading to post-ischemic neovascularization and tissue repair.
PMCID: PMC3703153  PMID: 22290850
Hematopoietic stem cell; Mobilization; Reactive oxygen species; Ischemia; Hypoxia
13.  HIF-1α regulates function and differentiation of myeloid-derived suppressor cells in the tumor microenvironment 
The Journal of Experimental Medicine  2010;207(11):2439-2453.
The hypoxic environment of tumors dictates the phenotype of local myeloid-derived suppressor cells (MDSCs) via HIF-1a expression; hypoxia converts splenic MDSCs from specific into nonspecific suppressors.
Myeloid-derived suppressor cells (MDSCs) are a major component of the immune-suppressive network described in cancer and many other pathological conditions. We demonstrate that although MDSCs from peripheral lymphoid organs and the tumor site share similar phenotype and morphology, these cells display profound functional differences. MDSC from peripheral lymphoid organs suppressed antigen-specific CD8+ T cells but failed to inhibit nonspecific T cell function. In sharp contrast, tumor MDSC suppressed both antigen-specific and nonspecific T cell activity. The tumor microenvironment caused rapid and dramatic up-regulation of arginase I and inducible nitric oxide synthase in MDSC, which was accompanied by down-regulation of nicotinamide adenine dinucleotide phosphate–oxidase and reactive oxygen species in these cells. In contrast to MDSC from the spleen, MDSC from the tumor site rapidly differentiated into macrophages. Exposure of spleen MDSC to hypoxia resulted in the conversion of these cells to nonspecific suppressors and their preferential differentiation to macrophages. Hypoxia-inducible factor (HIF) 1α was found to be primarily responsible for the observed effects of the tumor microenvironment on MDSC differentiation and function. Thus, hypoxia via HIF-1α dramatically alters the function of MDSC in the tumor microenvironment and redirects their differentiation toward tumor-associated macrophages, hence providing a mechanistic link between different myeloid suppressive cells in the tumor microenvironment.
PMCID: PMC2964584  PMID: 20876310
14.  Tracking Hypoxic Signaling within Encapsulated Cell Aggregates 
In Diabetes mellitus type 1, autoimmune destruction of the pancreatic β-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL.
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1α for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.
PMCID: PMC3369657  PMID: 22215075
Bioengineering;  Issue 58;  Cell encapsulation;  PEG;  cell aggregation;  hypoxia;  insulin secretion;  fluorescent imaging
15.  NADPH Oxidase Subunit 4-Mediated Reactive Oxygen Species Contribute to Cycling Hypoxia-Promoted Tumor Progression in Glioblastoma Multiforme 
PLoS ONE  2011;6(9):e23945.
Cycling and chronic tumor hypoxia are involved in tumor development and growth. However, the impact of cycling hypoxia and its molecular mechanism on glioblastoma multiforme (GBM) progression remain unclear.
Glioblastoma cell lines, GBM8401 and U87, and their xenografts were exposed to cycling hypoxic stress in vitro and in vivo. Reactive oxygen species (ROS) production in glioblastoma cells and xenografts was assayed by in vitro ROS analysis and in vivo molecular imaging studies. NADPH oxidase subunit 4 (Nox4) RNAi-knockdown technology was utilized to study the role of Nox4 in cycling hypoxia-mediated ROS production and tumor progression. Furthermore, glioblastoma cells were stably transfected with a retroviral vector bearing a dual reporter gene cassette that allowed for dynamic monitoring of HIF-1 signal transduction and tumor cell growth in vitro and in vivo, using optical and nuclear imaging. Tempol, an antioxidant compound, was used to investigate the impact of ROS on cycling hypoxia-mediated HIF-1 activation and tumor progression.
Principal Findings
Glioblastoma cells and xenografts were compared under cycling hypoxic and normoxic conditions; upregulation of NOX4 expression and ROS levels were observed under cycling hypoxia in glioblastoma cells and xenografts, concomitant with increased tumor cell growth in vitro and in vivo. However, knockdown of Nox4 inhibited these effects. Moreover, in vivo molecular imaging studies demonstrated that Tempol is a good antioxidant compound for inhibiting cycling hypoxia-mediated ROS production, HIF-1 activation, and tumor growth. Immunofluorescence imaging and flow cytometric analysis for NOX4, HIF-1 activation, and Hoechst 3342 in glioblastoma also revealed high localized NOX4 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion within the endogenous solid tumor microenvironment.
Cycling hypoxia-induced ROS via Nox4 is a critical aspect of cancer biology to consider for therapeutic targeting of cycling hypoxia-promoted HIF-1 activation and tumor progression in GBM.
PMCID: PMC3174133  PMID: 21935366
16.  Multiple Oxygen Tension Environments Reveal Diverse Patterns of Transcriptional Regulation in Primary Astrocytes 
PLoS ONE  2011;6(6):e21638.
The central nervous system normally functions at O2 levels which would be regarded as hypoxic by most other tissues. However, most in vitro studies of neurons and astrocytes are conducted under hyperoxic conditions without consideration of O2-dependent cellular adaptation. We analyzed the reactivity of astrocytes to 1, 4 and 9% O2 tensions compared to the cell culture standard of 20% O2, to investigate their ability to sense and translate this O2 information to transcriptional activity. Variance of ambient O2 tension for rat astrocytes resulted in profound changes in ribosomal activity, cytoskeletal and energy-regulatory mechanisms and cytokine-related signaling. Clustering of transcriptional regulation patterns revealed four distinct response pattern groups that directionally pivoted around the 4% O2 tension, or demonstrated coherent ascending/decreasing gene expression patterns in response to diverse oxygen tensions. Immune response and cell cycle/cancer-related signaling pathway transcriptomic subsets were significantly activated with increasing hypoxia, whilst hemostatic and cardiovascular signaling mechanisms were attenuated with increasing hypoxia. Our data indicate that variant O2 tensions induce specific and physiologically-focused transcript regulation patterns that may underpin important physiological mechanisms that connect higher neurological activity to astrocytic function and ambient oxygen environments. These strongly defined patterns demonstrate a strong bias for physiological transcript programs to pivot around the 4% O2 tension, while uni-modal programs that do not, appear more related to pathological actions. The functional interaction of these transcriptional ‘programs’ may serve to regulate the dynamic vascular responsivity of the central nervous system during periods of stress or heightened activity.
PMCID: PMC3124552  PMID: 21738745
17.  The p53 codon 72 proline allele is endowed with enhanced cell-death inducing potential in cancer cells exposed to hypoxia 
British Journal of Cancer  2007;96(8):1302-1308.
The preferential retention of the arginine allele at the p53 codon 72 locus is commonly observed in tumours from arginine/proline heterozygotes. Considering that cancer cells are harboured in a hypoxic environment in vivo, we here tested the hypothesis that the p53 codon 72 proline allele confers a survival disadvantage in presence of hypoxia. Here, we show that the transient transfection of the proline allele in p53 null cancer cells exposed to low oxygen tension or to the hypoxia-mimetic drug Desferoxamine induces a higher amount of cell death than the arginine allele. Accordingly, proline allele transiently transfected cell lines express lower levels of hypoxia pro-survival genes (HIF-1α, carbonic anhydrase IX, vascular endothelial growth factor, heme oxygenase-I, hepatocyte growth factor receptor, vascular endothelial growth factor receptor 2), compared to those transiently transfected with the arginine allele. Further, we report that the exposure of the arginine/proline heterozygote MCF-7 breast cancer cell line to cytotoxic concentration of Desferoxamine for several weeks, gives raise to hypoxia-resistant clones, carrying the arginine, but not the proline allele. These data indicate that the p53 codon 72 proline allele is less permissive for the growth of cancer cells in a hypoxic environment, and suggest that the preferential retention of the arginine allele in the tumour tissues of arginine/proline heterozygous patients may depend upon its lowered capacity to induce cell death in a hypoxic tumour environment.
PMCID: PMC2360160  PMID: 17406354
p53; codon 72; polymorphism; hypoxia
18.  Emerging evidence of the physiological role of hypoxia in mammary development and lactation 
Hypoxia is a physiological or pathological condition of a deficiency of oxygen supply in the body as a whole or within a tissue. During hypoxia, tissues undergo a series of physiological responses to defend themselves against a low oxygen supply, including increased angiogenesis, erythropoiesis, and glucose uptake. The effects of hypoxia are mainly mediated by hypoxia-inducible factor 1 (HIF-1), which is a heterodimeric transcription factor consisting of α and β subunits. HIF-1β is constantly expressed, whereas HIF-1α is degraded under normal oxygen conditions. Hypoxia stabilizes HIF-1α and the HIF complex, and HIF then translocates into the nucleus to initiate the expression of target genes. Hypoxia has been extensively studied for its role in promoting tumor progression, and emerging evidence also indicates that hypoxia may play important roles in physiological processes, including mammary development and lactation. The mammary gland exhibits an increasing metabolic rate from pregnancy to lactation to support mammary growth, lactogenesis, and lactation. This process requires increasing amounts of oxygen consumption and results in localized chronic hypoxia as confirmed by the binding of the hypoxia marker pimonidazole HCl in mouse mammary gland. We hypothesized that this hypoxic condition promotes mammary development and lactation, a hypothesis that is supported by the following several lines of evidence: i) Mice with an HIF-1α deletion selective for the mammary gland have impaired mammary differentiation and lipid secretion, resulting in lactation failure and striking changes in milk compositions; ii) We recently observed that hypoxia significantly induces HIF-1α-dependent glucose uptake and GLUT1 expression in mammary epithelial cells, which may be responsible for the dramatic increases in glucose uptake and GLUT1 expression in the mammary gland during the transition period from late pregnancy to early lactation; and iii) Hypoxia and HIF-1α increase the phosphorylation of signal transducers and activators of transcription 5a (STAT5a) in mammary epithelial cells, whereas STAT5 phosphorylation plays important roles in the regulation of milk protein gene expression and mammary development. Based on these observations, hypoxia effects emerge as a new frontier for studying the regulation of mammary development and lactation.
PMCID: PMC3929241  PMID: 24444333
Glucose transporter; Hypoxia; Hypoxia inducible factor; Lactation; Mammary development; Metabolism
19.  Tracking Hypoxic Signaling in Encapsulated Stem Cells 
Oxygen is not only a nutrient but also an important signaling molecule whose concentration can influence the fate of stem cells. This study details the development of a marker of hypoxic signaling for use with encapsulated cells. Testing of the marker was performed with adipose-derived stem cells (ADSCs) in two-dimensional (2D) and 3D culture conditions in varied oxygen environments. The cells were genetically modified with our hypoxia marker, which produces a red fluorescent protein (DsRed-DR), under the control of a hypoxia-responsive element (HRE) trimer. For 3D culture, ADSCs were encapsulated in poly(ethylene glycol)–based hydrogels. The hypoxia marker (termed HRE DsRed-DR) is built on a recombinant adenovirus and ADSCs infected with the marker will display red fluorescence when hypoxic signaling is active. This marker was not designed to measure local oxygen concentration but rather to show how a cell perceives its local oxygen concentration. ADSCs cultured in both 2D and 3D were exposed to 20% or 1% oxygen environments for 96 h. In 2D at 20% O2, the marker signal was not observed during the study period. In 1% O2, the fluorescent signal was first observed at 24 h, with maximum prevalence observed at 96 h as 59%±3% cells expressed the marker. In 3D, the signal was observed in both 1% and 20% O2. The onset of signal in 1% O2 was observed at 4 h, reaching maximum prevalence at 96 h with 76%±4% cells expressing the marker. Interestingly, hypoxic signal was also observed in 20% O2, with 13%±3% cells showing positive marker signal after 96 h. The transcription factor subunit hypoxia inducible factor-1α was tracked in these cells over the same time period by immunostaining and western blot analysis. Immunostaining results in 2D correlated well with our marker at 72 h and 96 h, but 3D results did not correlate well. The western blotting results in 2D and 3D correlated well with the fluorescent marker. The HRE DsRed-DR virus can be used to track the onset of this response for encapsulated, mesenchymal stem cells. Due to the importance of hypoxic signaling in determination of stem cell differentiation, this marker could be a useful tool for the tissue engineering community.
PMCID: PMC4003466  PMID: 22250882
20.  Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. 
Molecular and Cellular Biology  1994;14(9):5997-6003.
Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.
PMCID: PMC359125  PMID: 8065332
21.  Theoretical analysis of the dose dependence of the oxygen enhancement ratio and its relevance for clinical applications 
The increased resistance of hypoxic cells to ionizing radiation is usually believed to be the primary reason for treatment failure in tumors with oxygen-deficient areas. This oxygen effect can be expressed quantitatively by the oxygen enhancement ratio (OER). Here we investigate theoretically the dependence of the OER on the applied local dose for different types of ionizing irradiation and discuss its importance for clinical applications in radiotherapy for two scenarios: small dose variations during hypoxia-based dose painting and larger dose changes introduced by altered fractionation schemes.
Using the widespread Alper-Howard-Flanders and standard linear-quadratic (LQ) models, OER calculations are performed for T1 human kidney and V79 Chinese hamster cells for various dose levels and various hypoxic oxygen partial pressures (pO2) between 0.01 and 20 mmHg as present in clinical situations in vivo. Our work comprises the analysis for both low linear energy transfer (LET) treatment with photons or protons and high-LET treatment with heavy ions. A detailed analysis of experimental data from the literature with respect to the dose dependence of the oxygen effect is performed, revealing controversial opinions whether the OER increases, decreases or stays constant with dose.
The behavior of the OER with dose per fraction depends primarily on the ratios of the LQ parameters alpha and beta under hypoxic and aerobic conditions, which themselves depend on LET, pO2 and the cell or tissue type. According to our calculations, the OER variations with dose in vivo for low-LET treatments are moderate, with changes in the OER up to 11% for dose painting (1 or 3 Gy per fraction compared to 2 Gy) and up to 22% in hyper-/hypofractionation (0.5 or 20 Gy per fraction compared to 2 Gy) for oxygen tensions between 0.2 and 20 mmHg typically measured clinically in hypoxic tumors. For extremely hypoxic cells (0.01 mmHg), the dose dependence of the OER becomes more pronounced (up to 36%). For high LET, OER variations up to 4% for the whole range of oxygen tensions between 0.01 and 20 mmHg were found, which were much smaller than for low LET.
The formalism presented in this paper can be used for various tissue and radiation types to estimate OER variations with dose and help to decide in clinical practice whether some dose changes in dose painting or in fractionation can bring more benefit in terms of the OER in the treatment of a specific hypoxic tumor.
PMCID: PMC3283483  PMID: 22172079
Oxygen enhancement ratio; hypoxia; oxygen effect; fractionation; high LET; radiation therapy
22.  Imaging and Targeting of the Hypoxia-inducible Factor 1-active Microenvironment 
Journal of Toxicologic Pathology  2009;22(2):93-100.
Human solid tumors contain hypoxic regions that have considerably lower oxygen tension than normal tissues. They are refractory to radiotherapy and anticancer chemotherapy. Although more than half a century has passed since it was suggested that tumour hypoxia correlates with poor treatment outcomes and contributes to recurrence of cancer, no fundamental solution to this problem has been found. Hypoxia-inducible factor-1(HIF-1) is the main transcription factor that regulates the cellular response to hypoxia. It induces various genes, whose function is strongly associated with malignant alteration of the entire tumour. The cellular changes induced by HIF-1 are extremely important therapeutic targets of cancer therapy, particularly in therapy against refractory cancers. Therefore, targeting strategies to overcome the HIF-1-active microenvironment are important for cancer therapy. To Target HIF-1-active/ hypoxic tumor cells, we developed a fusion protein drug, PTD-ODD-Procaspase-3 that selectively induces cell death in HIF-1-active/hypoxic cells. The drug consists of the following three functional domains: the protein transduction domain (PTD), which efficiently delivers the fusion protein to hypoxic tumor cells, the ODD domain, which has a VHL-mediated protein destruction motif of human HIF-1α protein and confers hypoxia-dependent stabilization to the fusion proteins, and the human procaspase-3 proenzyme responsible for the cytocidal activity of the protein drug. In vivo imaging systems capable of monitoring HIF-1 activity in transplanted human cancer cells in mice are useful in evaluating the efficiency of these drugs and in study of HIF-1-active tumor cells.
PMCID: PMC3246054  PMID: 22271982
hypoxia-inducible factor 1 (HIF-1); tumour hypoxia; hypoxia responsive element (HRE); protein transduction domain (PTD); bioluminescence; in vivo imaging
23.  Hypoxic regulation of cytoglobin and neuroglobin expression in human normal and tumor tissues 
Cytoglobin (Cygb) and neuroglobin (Ngb) are recently identified globin molecules that are expressed in vertebrate tissues. Upregulation of Cygb and Ngb under hypoxic and/or ischemic conditions in vitro and in vivo increases cell survival, suggesting possible protective roles through prevention of oxidative damage. We have previously shown that Ngb is expressed in human glioblastoma multiforme (GBM) cell lines, and that expression of its transcript and protein can be significantly increased after exposure to physiologically relevant levels of hypoxia. In this study, we extended this work to determine whether Cygb is also expressed in GBM cells, and whether its expression is enhanced under hypoxic conditions. We also compared Cygb and Ngb expression in human primary tumor specimens, including brain tumors, as well as in human normal tissues. Immunoreactivity of carbonic anhydrase IX (CA IX), a hypoxia-inducible metalloenzyme that catalyzes the hydration of CO2 to bicarbonate, was used as an endogenous marker of hypoxia.
Cygb transcript and protein were expressed in human GBM cells, and this expression was significantly increased in most cells following 48 h incubation under hypoxia. We also showed that Cygb and Ngb are expressed in both normal tissues and human primary cancers, including GBM. Among normal tissues, Cygb and Ngb expression was restricted to distinct cell types and was especially prominent in ductal cells. Additionally, certain normal organs (e.g. stomach fundus, small bowel) showed distinct regional co-localization of Ngb, Cygb and CA IX. In most tumors, Ngb immunoreactivity was significantly greater than that of Cygb. In keeping with previous in vitro results, tumor regions that were positively stained for CA IX were also positive for Ngb and Cygb, suggesting that hypoxic upregulation of Ngb and Cygb also occurs in vivo.
Our finding of hypoxic up-regulation of Cygb/Ngb in GBM cell lines and human tumor tissues suggests that these globin molecules may be part of the repertoire of defense mechanisms that allow cancer cells to survive in hypoxic microenvironments.
PMCID: PMC2945342  PMID: 20828399
24.  The Clinical Importance of Assessing Tumor Hypoxia: Relationship of Tumor Hypoxia to Prognosis and Therapeutic Opportunities 
Antioxidants & Redox Signaling  2014;21(10):1516-1554.
Tumor hypoxia is a well-established biological phenomenon that affects the curability of solid tumors, regardless of treatment modality. Especially for head and neck cancer patients, tumor hypoxia is linked to poor patient outcomes. Given the biological problems associated with tumor hypoxia, the goal for clinicians has been to identify moderately to severely hypoxic tumors for differential treatment strategies. The “gold standard” for detecting and characterizing of tumor hypoxia are the invasive polarographic electrodes. Several less invasive hypoxia assessment techniques have also shown promise for hypoxia assessment. The widespread incorporation of hypoxia information in clinical tumor assessment is severely impeded by several factors, including regulatory hurdles and unclear correlation with potential treatment decisions. There is now an acute need for approved diagnostic technologies for determining the hypoxia status of cancer lesions, as it would enable clinical development of personalized, hypoxia-based therapies, which will ultimately improve outcomes. A number of different techniques for assessing tumor hypoxia have evolved to replace polarographic pO2 measurements for assessing tumor hypoxia. Several of these modalities, either individually or in combination with other imaging techniques, provide functional and physiological information of tumor hypoxia that can significantly improve the course of treatment. The assessment of tumor hypoxia will be valuable to radiation oncologists, surgeons, and biotechnology and pharmaceutical companies who are engaged in developing hypoxia-based therapies or treatment strategies. Antioxid. Redox Signal. 21, 1516–1554.
I. Introduction
II. The Clinical Importance of Tumor Hypoxia
A. Pathophysiology of hypoxia
B. Hypoxia's negative impact on the effectiveness of curative treatment
1. Hypoxic tumors accumulate and propagate cancer stem cells
2. Hypoxia reduces the effectiveness of radiotherapy
3. Hypoxia increases metastasis risk and reduces the effectiveness of surgery
4. Hypoxic tumors are resistant to the effects of chemotherapy and chemoradiation
C. Hypoxia is prognostic for poor patient outcomes
III. Diagnosis of Tumor Hypoxia
A. Direct methods
1. Oxygen electrode—direct pO2 measurement most used in cancer research
2. Phosphorescence quenching—alternative direct pO2 measurement
3. Electron paramagnetic resonance
4. 19F-magnetic resonance spectroscopy
5. Overhauser-enhanced MRI
B. Endogenous markers of hypoxia
1. Hypoxia-inducible factor-1α
2. Carbonic anhydrase IX
3. Glucose transporter 1
4. Osteopontin
5. A combined IHC panel of protein markers for hypoxia
6. Comet assay
C. Physiologic methods
1. Near-infrared spectroscopy/tomography—widely used for pulse oximetry
2. Photoacoustic tomography
3. Contrast-enhanced color duplex sonography
4. MRI-based measurements
5. Blood oxygen level-dependent MRI
6. Pimonidazole
7. EF5 (pentafluorinated etanidazole)
8. Hypoxia PET imaging—physiologic hypoxia measurement providing tomographic information
a. 18F-fluoromisonidazole
b. 18F-fluoroazomycinarabinofuranoside
c. 18F-EF5 (pentafluorinated etanidazole)
d. 18F-flortanidazole
e. Copper (II) (diacetyl-bis (N4-methylthiosemicarbazone))
f. 18F-FDG imaging of hypoxia
IV. Modifying Hypoxia to Improve Therapeutic Outcomes
A. Use of hypoxia information in radiation therapy planning
B. Use of hypoxia assessment for selection of patients responsive to nimorazole
C. Use of hypoxia assessment for selection of patients responsive to tirapazamine
D. Use of hypoxia assessment for selection of patients responsive to oxygen delivery therapies
V. Concluding Remarks
PMCID: PMC4159937  PMID: 24512032
25.  Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxia-inducible factor-1 
Placental hypoxia has been implicated in pregnancy pathologies, including fetal growth restriction and preeclampsia; however, the mechanism by which the trophoblast cell responds to hypoxia has not been adequately explored. Glucose transport, a process crucial to fetoplacental growth, is upregulated by hypoxia in a number of cell types. We investigated the effects of hypoxia on the regulation of trophoblast glucose transporter (GLUT) expression and activity in BeWo choriocarcinoma cells, a trophoblast cell model, and human placental villous tissue explants. GLUT1 expression in BeWo cells was upregulated by the hypoxia-inducing chemical agents desferroxamine and cobalt chloride. Reductions in oxygen tension resulted in dose-dependent increases in GLUT1 and GLUT3 expression. Exposure of cells to hypoxic conditions also resulted in an increase in transepithelial glucose transport. A role for hypoxia-inducible factor (HIF)-1 was suggested by the increase in HIF-1α as a result of hypoxia and by the increase in GLUT1 expression following treatment of BeWo with MG-132, a proteasomal inhibitor that increases HIF-1 levels. The function of HIF-1 was confirmed in experiments where the hypoxic upregulation of GLUT1 and GLUT3 was inhibited by antisense HIF-1α. In contrast to BeWo cells, hypoxia produced minimal increases in GLUT1 expression in explants; however, treatment with MG-132 did upregulate syncytial basal membrane GLUT1. Our results show that GLUTs are upregulated by hypoxia via a HIF-1-mediated pathway in trophoblast cells and suggest that the GLUT response to hypoxia in vivo will be determined not only by low oxygen tension but also by other factors that modulate HIF-1 levels.
PMCID: PMC4497554  PMID: 17442736
glucose transporter 1; glucose transporter 3; glucose transport

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