Hypoxia-induced Changes Research Articles

Evolution of physiological performance capacities and environmental adaptation: insights from high-elevation deer mice (Peromyscus maniculatus).

Analysis of variation in whole-animal performance can shed light on causal connections between specific traits, integrated physiological capacities, and Darwinian fitness. Here, we review and synthesize information on naturally occurring variation in physiological performance capacities and how it relates to environmental adaptation in deer mice (Peromyscus maniculatus). We discuss how evolved changes in aerobic exercise capacity and thermogenic capacity have contributed to adaptation to high elevations. Comparative work on deer mice at high and low elevations has revealed evolved differences in aerobic performance capacities in hypoxia. Highland deer mice have consistently higher aerobic performance capacities under hypoxia relative to lowland natives, consistent with the idea that it is beneficial to have a higher maximal metabolic rate (as measured by the maximal rate of O2 consumption, VO2max) in an environment characterized by lower air temperatures and lower O2 availability. Observed differences in aerobic performance capacities between highland and lowland deer mice stem from changes in numerous subordinate traits that alter the flux capacity of the O2-transport system, the oxidative capacity of tissue mitochondria, and the relationship between O2 consumption and ATP synthesis. Many such changes in physiological phenotype are associated with hypoxia-induced changes in gene expression. Research on natural variation in whole-animal performance forms a nexus between physiological ecology and evolutionary biology that requires insight into the natural history of the study species.

Spermidine Prevents Heart Injury in Neonatal Rats Exposed to Intrauterine Hypoxia by Inhibiting Oxidative Stress and Mitochondrial Fragmentation.

Intrauterine hypoxia (IUH) is a common intrauterine dysplasia that can cause programming of the offspring cardiovascular system. In this study, we hypothesized that placental treatment with spermidine (SPD) can prevent heart injury in neonatal offspring exposed to IUH. Pregnant rats were exposed to 21% O2 or 10% O2 (hypoxia) for 7 days prior to term or were exposed to hypoxia and intraperitoneally administered SPD or SPD+difluromethylornithine (DFMO) on gestational days 15-21. Seven-day-old offspring were then sacrificed to assess several parameters. Our results demonstrated that IUH led to decreased myocardial ornithine decarboxylase (ODC) and increased spermidine/spermine N1-acetyltransferase (SSAT) expression in the offspring. IUH also resulted in decreased offspring body weight, heart weight, cardiomyocyte proliferation, and antioxidant capacity and increased cardiomyocyte apoptosis and fibrosis. Furthermore, IUH caused mitochondrial structure abnormality, dysfunction, and decreased biogenesis and led to a fission/fusion imbalance in offspring hearts. In vitro, hypoxia induced mitochondrial ROS accumulation, decreased membrane potential, and increased fragmentation. Notably, all hypoxia-induced changes analyzed in this study were prevented by SPD. Thus, in utero SPD treatment is a potential strategy for preventing IUH-induced neonatal cardiac injury.

Impairment of HIF-1α-mediated metabolic adaption by NRF2-silencing in breast cancer cells

Hypoxia, a common element in the tumor environment, leads to Hypoxia-Inducible Factor-1α (HIF-1α) stabilization to modulate cellular metabolism as an adaptive response. In a previous study, we showed that inhibition of the nuclear factor erythroid 2-like-2 (NFE2L2; NRF2), a master regulator of many genes coping with electrophilic and oxidative stress, elevated the level of miR-181c and induced mitochondrial dysfunction in colon cancer cells. In this study, we demonstrate that NRF2-silencing hindered HIF-1α accumulation in hypoxic breast cancer cells and subsequently suppressed hypoxia-inducible expression of glycolysis-associated glucose transporter-1, hexokinase-2, pyruvate dehydrogenase kinase-1, and lactate dehydrogenase A. HIF-1α dysregulation in NRF2-silenced cancer cells was associated with miR-181c elevation. Overexpression of miR-181c in breast cancer cells blocked HIF-1α accumulation and diminished hypoxia-inducible levels of glycolysis enzymes, whereas the inhibition of miR-181c in NRF2-silenced cells restored HIF-1α accumulation. In a subsequent metabolomic analysis, hypoxic incubation increased the levels of metabolites involved in glycolysis and activated the pentose phosphate pathway (PPP) in control cells. However, these elevations were less pronounced in NRF2-silenced cells. In particular, hypoxic incubation increased the levels of amino acids, which implies a shift to catabolic metabolism, and the increased levels were higher in control cells than in NRF2-silenced cells. Concurrently, hypoxia activated BCL2 interacting protein 3 (BNIP3)-mediated autophagy in the control cells and miR-181c was found to be involved in this autophagy activation. Taken together, these results show that hypoxia-induced metabolic changes to glycolysis, the PPP, and autophagy are inhibited by NRF2-silencing through miR-181c-mediated HIF-1α dysregulation. Therefore, targeting NRF2/miR-181c could be an effective strategy to counteract HIF-1α-orchestrated metabolic adaptation of hypoxic cancer cells.

The Effects of Remote Ischemic Preconditioning on Cognitive Function and Exercise Hyperemia in Isocapnic Hypoxia

Impairments in neurological and skeletal muscle function can occur in conditions of decreased oxygen delivery and vascular dysfunction can exacerbate any impairments. Remote ischemic preconditioning (RIPC) has been used to improve vascular functioning during exercise and cognitive tasks, but this intervention has yet to be tested in hypoxic conditions. Here, we test the hypothesis that RIPC can attenuate the hypoxia‐induced decline in cognitive function and also impact forearm blood flow during hypoxic exercise. Healthy, young men and women participated in two counterbalanced experimental sessions, consisting of two trials of rhythmic dynamic handgrip exercise (4 min, 10% maximal voluntary contraction) followed by computerized cognitive testing, once in normoxia (21% inspired O2), and once in isocapnic hypoxia (target O2 saturation ~85% via pulse oximetry). The cognitive tests (CNS Vital Signs) measured composite memory, verbal memory, visual memory, and processing speed. During rest and handgrip exercise, forearm blood flow (FBF) responses were determined via Doppler ultrasound and muscle oxygenation was assessed using near infrared spectroscopy (Moxy). Systemic hemodynamics [heart rate (ECG) and non‐invasive beat‐to‐beat blood pressure (Finometer)] were collected throughout. One experimental session consisted of RIPC (4 × 5 min thigh cuff inflation; 200 mmHg) between the normoxic and hypoxic trials whereas the other was a sham (~10 mmHg cuff pressure) intervention. Percent change (%Δ) from normoxia to hypoxia was determined for cognitive test scores. Preliminary results (n=6, mean±sem) demonstrate modest hypoxia‐induced declines in composite, verbal, and visual memory (−8.6±4.4%, −11.2±6.6%, −5.2±3.8%) but not processing speed in sham conditions. The RIPC intervention had no consistent impact on these hypoxia‐induced changes. Exercise hyperemia was increased in hypoxia vs normoxia (177±12 vs 163±6 ml/min in hypoxia vs normoxia, respectively) and RIPC modestly increased exercise hyperemia in hypoxia (191±10 ml/min) but had little effect during normoxia (167±12 ml/min). We conclude that RIPC has a minimal effect on the impact of hypoxia on cognitive function and can augment hypoxic exercise hyperemia in young, healthy humans.Support or Funding InformationUniversity of Dayton University Honors Program, Berry Summer Thesis Institute (JNS)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Translational and transcriptional responses in human primary hepatocytes under hypoxia.

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes. NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.

Liver CEBPβ Modulates the Kynurenine Metabolism and Mediates the Motility for Hypoxia-Induced Central Fatigue in Mice.

Central fatigue is defined as a failure of the central nervous system to adequately drive the muscle, manifesting limited development, and maintenance of locomotor activity. A plateau in hypoxia leads to central fatigue and followed by maximal motility recession. However, the underlying mechanism is still unclear. The present study describes a mechanism by which liver CEBPβ (CCAAT/enhancer-binding protein beta) induced by hypoxic environment alters the kynurenine (KYN) metabolism and causes the suppression of motility function recession. The activation of CEBPβ under hypoxia increases the liver expression of tryptophan dioxygenase, thereby enhancing the conversion of tryptophan into KYN; the KYN metabolite can traverse the blood-brain barrier and result in the suppression of motility function. However, the knockdown of CEBPβ by injecting pAAV-shRNA-CEBPβ via the hepatic portal vein reduces the KYN production and improves the motility function. KYN is a neurochemical that which restricts the exercise capacity after injection in the basal ganglia in mice. Reducing the plasma KYN protects the brain from hypoxia-induced changes associated with fatigue, and the knockdown liver of CEBPβ in mice renders resistance to fatigue post-acute hypoxia or tryptophan treatment. This study reveals resistance to central fatigue as a strategy for acclimatization to hypoxia mediated by transcription factor CEBPβ in the liver.

Integrative Omic Profiling Reveals Unique Hypoxia Induced Signatures in Gastric Cancer Associated Myofibroblasts.

Although hypoxia is known to contribute to several aspects of tumour progression, relatively little is known about the effects of hypoxia on cancer-associated myofibroblasts (CAMs), or the consequences that conditional changes in CAM function may have on tumour development and metastasis. To investigate this issue in the context of gastric cancer, a comparative multiomic analysis was performed on populations of patient-derived myofibroblasts, cultured under normoxic or hypoxic conditions. Data from this study reveal a novel set of CAM-specific hypoxia-induced changes in gene expression and secreted proteins. Significantly, these signatures are not observed in either patient matched adjacent tissue myofibroblasts (ATMs) or non-cancer associated normal tissue myofibroblasts (NTMs). Functional characterisation of different myofibroblast populations shows that hypoxia-induced changes in gene expression not only enhance the ability of CAMs to induce cancer cell migration, but also confer pro-tumorigenic (CAM-like) properties in NTMs. This study provides the first global mechanistic insight into the molecular changes that contribute to hypoxia-induced pro-tumorigenic changes in gastric stromal myofibroblasts.

FGF21 Protects Against Hypoxia Injury Through Inducing HSP72 in Cerebral Microvascular Endothelial Cells.

Background: Fibroblast growth factor 21 (FGF21), a member of a family of atypical FGFs, functions as cytokine to control endocrinology and metabolism. Recently, the roles of FGF21 in cardio-cerebral-vascular diseases have been gradually uncovered. In the present study, we investigated the effect of FGF21 on bEnd.3 cerebral microvascular endothelial cells (CMECs) upon hypoxia stress. Methods and Results: CMECs were cultured in the condition of 1% O2 for 8 h to induce hypoxia stimuli. For FGF21 treatment, recombinant FGF21 (50 nM) was added into the culture medium. Various biomedical assays were used to evaluate the hypoxia-induced injury in CMECs. Under normoxia condition, FGF21 had no obvious effect on cell viability and did not cause any cytotoxicity on CMECs. Under hypoxia condition, FGF21 significantly attenuated the hypoxia-induced injury, evidenced by the influences of FGF21 on CMEC viability and LDH release. TUNEL staining assay and immunoblotting of caspase-3 showed that FGF21 reduced hypoxia-induced apoptosis in CMECs. Mechanistically, FGF21 treatment compromised the hypoxia-induced changes of reactive oxygen species, malondialdehyde, total antioxidant activity, and total superoxide dismutase levels. FGF21 administration decreased hypoxia-induced matrix metalloprotein 3 and matrix metalloprotein 2/9 activity in CMECs. Activities of cyclooxygenase-2 and NF-κB-p65, two pro-inflammatory factors, were also upregulated by hypoxia but suppressed by FGF21. At last, we found that FGF21 increased heat shock protein family A member 1A (HSP72) mRNA and protein expression. Blockade of HSP72 by a pharmacological inhibitor VER155008 or specific siRNA-mediated knockdown abrogated the protection of FGF21 against hypoxia in CMECs. Conclusion: These data demonstrate that FGF21 protects against hypoxia stress-induced injury in CMECs by inducing HSP72 expression, suggesting a therapeutic value of FGF21 in hypoxia-related brain diseases such as ischemic stroke and acute mountain sickness.

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation

BackgroundA hypoxic microenvironment leads to an increase in the invasiveness and the metastatic potential of cancer cells within tumors via the epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. However, hypoxia-induced changes in the expression and function of candidate stem cell markers and their possible molecular mechanism is still not understood.MethodsLung cell lines were analyzed in normoxic or hypoxic conditions. For screening among the stem cell markers, a transcriptome analysis using next-generation sequencing was performed. For validation, the EMT and stem cell characteristics were analyzed. To determine whether an epigenetic mechanism was involved, the cell lines were treated with a DNA methyltransferase inhibitor (AZA), and methylation-specific PCR and bisulfite sequencing were performed.ResultsNext-generation sequencing revealed that the CXCR4 expression was significantly higher after the hypoxic condition, which functionally resulted in the EMT and cancer stemness acquisition. The acquisition of the EMT and stemness properties was inhibited by treatment with CXCR4 siRNA. The CXCR4 was activated by either the hypoxic condition or treatment with AZA. The methylation-specific PCR and bisulfite sequencing displayed a decreased CXCR4 promoter methylation in the hypoxic condition.ConclusionsThese results suggest that hypoxia-induced acquisition of cancer stem cell characteristics was associated with CXCR4 activation by its aberrant promoter demethylation.

Hypoxia-induced changes in survival, immune response and antioxidant status of the Pacific white shrimp (Litopenaeus vannamei ) fed with graded levels of dietary myo -inositol

Hypoxia-induced changes in survival, immune response and antioxidant status of the Pacific white shrimp (<i>Litopenaeus vannamei</i> ) fed with graded levels of dietary <i>myo</i> -inositol

Arabidopsis PLDs with C2-domain function distinctively in hypoxia.

Hypoxia (oxygen deprivation) causes metabolic disturbances at physiological, biochemical and genetic levels and results in decreased plant growth and development. Phospholipase D (PLD)-mediated signaling was reported for abiotic and biotic stress signaling events in plants. To investigate the participatory role of PLDs also in hypoxia signaling, we used wild type of Arabidopsis thaliana and 10 pld isoform mutants containing C2-domain. Hypoxia-induced changes in three major signaling players, namely, cytosolic free calcium (Ca2+ cyt ), reactive oxygen species (ROS) and phosphatidic acid (PA), were determined in mesophyll protoplasts. The Ca2+ cyt and ROS levels were monitored by fluorescence microscopy and confocal imaging, while PA levels were quantified by an enzymatic method. Our findings reveal that the elevations of cytosolic calcium and PA are reduced in all the 10 mutants dysfunctional in PLD isoforms. The hypoxia-related changes in both calcium and ROS show different kinetic patterns depending on the type of PLD studied. Pharmacological experiments confirm that both external and internal sources contribute to calcium and ROS accumulation under hypoxia. PLDα1-3, PLDβ1 and PLDγ1-3 are likely involved in calcium signaling under hypoxia as well as in PA production, while all investigated PLDs, except for PLDγ3, take part in ROS elevation.

Colorectal Cancer Cell Line SW480 and SW620 Released Extravascular Vesicles: Focus on Hypoxia-induced Surface Proteome Changes

Extravascular vesicle (EV) proteome closely reflects the proteome of the cell of origin. Therefore, cancer cell-derived EV proteomic analysis could help in identifying cancer biomarkers. This study's goal was to investigate hypoxia-induced proteomic changes in EV released from hypoxic human isogenic non-metastatic colorectal cancer cells SW480 and metastatic colorectal cancer cells SW620. EV were characterized by western blot, transmission electron microscopy, proteomic analysis using liquid chromatography time-of-flight-mass spectrometry and quantified by an label-free intensity-based absolute quantitation (iBAQ) approach. A total of 16 proteins in hypoxic EV exceeded normoxic EV protein levels in SW480 EV. Of them, 15 were also found in EV of hypoxic SW620 cells. The expression levels of proteins differed quantitatively: iBAQ (log 10) scores of the levels of five proteins in SW620 EV exceeded those in hypoxic SW480 EV and levels of 11 proteins in SW480 EV exceeded those of SW620 EV. Under hypoxia, colorectal cancer cells release EV that qualitatively and quantitatively change the surface proteome. In the future, the specific hypoxia-induced proteins could be developed as new biomarkers for non-invasive assessment of tumour hypoxia.

Protective effects of dexamethasone on hypoxia-induced retinal edema in a mouse model

Hypoxia-induced retinal edema primarily induced by vascular lesion is seen in various conditions such as diabetic retinopathy (DR) and retinal vein occlusion (RVO). The edematous changes in these conditions occur mainly in intermediate and deep layers of retina as a result of disruption of the inner blood-retinal barrier (iBRB). However, the effect of direct and acute hypoxia on iBRB remains to be elucidated. To investigate direct and acute hypoxia-induced changes in retina, especially in astrocytes/Müller cells that are involved in the maintenance of retinal structure and function, we developed an adult mouse model of hypoxia-induced retinal edema by 24-h exposure in a 6% oxygen environment. Immunohistochemical staining of glial fibrillary acidic protein (GFAP) was enhanced mainly in the superficial layer of the hypoxic retina, corresponding to edematous change. Electron microscopic observation of the hypoxic retina showed vacuole formation in astrocyte/Müller cell foot processes around capillaries in the superficial layer, while no abnormal findings in the perivascular areas were found in intermediate and deep layers. Increase in vascular leakage quantified by Evans blue dye and tight junction breakdown detected by electron-dense tracer were observed in the hypoxia group. In the hypoxic retina, microglia was activated and relative gene expressions of pro-inflammatory cytokines were significantly upregulated. Dexamethasone suppressed these hypoxia-induced pathological reactions. Thus, unlike DR and RVO that induce iBRB breakdown in deeper retinal layers, atmospheric hypoxia induced iBRB disruption with subsequent edematous change mainly in the superficial layer of the retina, and that dexamethasone prevented these pathological changes. In this mouse model, direct and acute hypoxia induces retinal edema in the superficial layer of the retina with morphological changes of astrocytes/Müller cells, and is potentially useful for ophthalmic research in the field related to retinal hypoxia and its treatment.

The effect of hypoxia on ZEB1 expression in a mimetic system of the blood-brain barrier

The blood-brain barrier consists of a tightly sealed monolayer of endothelial cells being vital in maintaining a stable intracerebral microenvironment. The barrier is receptive to leakage upon exposure to environmental factors, like hypoxia, and its disruption has been suggested as a constituent in the pathophysiology of both neurological and psychiatric disorders. The schizophrenia associated ZEB1 gene encodes a transcription factor susceptible to transcriptional control by a hypoxia induced factor, HIF1A, known to be implicated in blood-brain barrier dysfunction. However, whether ZEB1 is also implicated in maintaining blood-brain barrier integrity upon hypoxia is unknown. Here we assessed Hif1a, Zo1 and Zeb1 mRNA expression and ZO1 protein abundancy in a mimetic system of the in vivo blood-brain barrier comprising mouse brain endothelial cells subjected to the norm- and proven hypoxic conditions. Despite that, Hif1a mRNA expression was significantly increased, clearly indicating that the oxygen-deprived environment introduced a hypoxia response in the cells, we found no hypoxia-induced changes in neither ZO1 abundancy nor in the expression of Zo1 and Zeb1 mRNA. However, independent of hypoxia status, we found that Zeb1 and Zo1 mRNA expression is highly correlated. Further studies are warranted that investigate the implication of the ZEB1/ZO1 axis in blood-brain barrier maintenance under different physiological conditions.

Hypoxia promotes acquisition of aggressive phenotypes in human malignant mesothelioma

BackgroundHypoxia is a hallmark of the solid tumor microenvironment and is associated with poor outcomes in cancer patients. The present study was performed to investigate mechanisms underlying the hypoxia-induced phenotypic changes using human malignant mesothelioma (HMM) cells.MethodsHypoxic conditions were achieved by incubating HMM cells in the air chamber. The effect of hypoxia on phenotype changes in HMM cells was investigated by performing in vitro clonogenicity, drug resistance, migration, and invasion assays. Signaling pathways and molecules involved in the more aggressive behaviors of HMM cells under hypoxia were investigated. A two-tailed unpaired Student’s t-test or one-way ANOVA with Bonferroni post-test correction was used in this study.ResultsHypoxic conditions upregulated hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α in parallel with the upregulation of its target, Glut-1, in HMM cells. In vitro clonogenicity of HMM cells was significantly increased in hypoxic conditions, but the proliferation of cells at a high density in hypoxia was lower than that in normoxic conditions. The expression levels of HIF-2α and Oct4 were increased in hypoxic HMM cells. The percentage of cells with high CD44 expression was significantly higher in HMM cells cultured in hypoxia than those cultured in normoxia. Hypoxia significantly enhanced the resistance of HMM cells to cisplatin, which occurred through cytoprotection against cisplatin-induced apoptosis. While cisplatin treatment decreased the ratio of Bcl-2 to Bax in normoxic condition, hypoxia conversely increased the ratio in HMM cells treated with cisplatin. Hypoxia increased the mobility and invasiveness of HMM cells. Epithelial to mesenchymal transition was promoted, which was indicated by the repression of E-cadherin and the concomitant increase of vimentin in HMM cells.ConclusionsThe data illustrated that hypoxic conditions augmented the aggressive phenotypes of HMM cells at the biological and molecular levels. The present study provides valuable background information beginning to understand aggressiveness of HMM in tumor microenvironments, suggesting that a control measure for tumor hypoxia may be an effective therapeutic strategy to reduce the aggressiveness of cancer cells in HMM patients.

Human Amniotic Epithelial Cells Integrated Into the Islet Heterospheroids Enhance Insulin Secretion and Protect Islet Cells from Hypoxic Injury

Background Recent advancements in tissue engineering shows that generating multicellular spheroids by combining different cell types can enhance their regenerative capacity. Human amniotic epithelial cells (hAECs) gained great interest in regenerative medicine due to their availability, safety, regenerative, immunomodulatory and anti-inflammatory properties. The aim of this study was to determine whether combination of hAECs with islet cells in the same spheroid would further improve islet cell survival and function in vitro under normoxic and hypoxic conditions. Methods Functional Islet spheroids were generated on 3D agarose-patterned microwells. To form homospheroids dispersed rat islet cells (ICs) and hAECs (128,000 cells/mold and 500cell/spheroid) were seeded alone. Heterospheroids were formed by mixing ICs and hAECs at ratio of 1:1. Engineered islet homo- and heterospheroids were cultured under normoxic and hypoxic conditions for 16 h. For all conditions, cell viability, GSIS, total islet cellular insulin content were detected. Hypoxia-induced changes in gene expression were assessed by real time PCR analysis. Next we considered possible molecular mechanisms behind the beneficial effect of hAECs on islet cell function. For this purpose, the mRNA expression levels of Hif-1α, Casp3, Casp8, Casp9 and Bcl2 were determined. Results Confocal laser scanning microscopy showed uniform distribution of islet cells and hAECs throughout of hybrid spheroids, without evidence of cell loss. Quantifications of the insulin positive area ratios to nuclei, showed that heterospheroids expressed more insulin compared to homospheroids. Moreover, islet heterospheroids expressed significantly more E-cadherin, key cell-to-cell adhesion molecule then homospheroids. The stimulation index of the heterospheroids was significantly higher than those of homospheroids. Exposure to hypoxia rapidly caused fragmentation of homospheroids and augmented cell membrane permeability. In contrary considerably less dead signals were observed within heterospheroids. As anticipated, glucose responsiveness of homospheroids was seriously impaired; the average glucose stimulation index of the heterospheroids was significantly higher than that of the homospheroids (1.7±0.4 vs 0.9±0.5, p < 0.05). This was correlated with downregulation of apoptotic genes Casp3, Casp8 and Casp9 and 2 fold upregulation of antiapoptotic gene Bcl2. Conclusions These data indicate that incorporation of hAECs into the islet heterospheroids improves the secretory function and viability of islet cells both in conventional culture and in hypoxic conditions.

PO-224 Fourier transform infrared spectroscopy-based detection of hypoxia-induced biomolecular changes in different cancer cell lines

IntroductionOur previous studies have shown that under hypoxic stress human colorectal and breast cancer cells produce higher amounts of lipids compared to that under normoxic environment. The aim of this study was to estimate hypoxia induced cell metabolic response via the biochemical composition in prostate and lung cancer cells by using Fourier transform infrared spectroscopy (FTIR).Material and methodsLung cancer cell lines NCI-H69, COR-L23 and prostate cancer cell line PC3, and primary prostate cancer culture NFI-56 were seed in serum-free medium in density 1 × 106 per well in triplicates in 6-well plates and incubated for 48 in normoxic and hypoxic (5% CO2, 1% O2 and 94% N2) conditions. Cells were washed and snap frozen within 5 min after incubation. Hypoxic response in cells was verified by HIF1α expression in Western blot analysis. FTIR spectra of cancer cells were registered using HTS-XT microplate reader coupled with Vertex 70 (Bruker), and recorded in the frequency range of 4000–600 cm−1, with the spectral resolution of 4 cm−1 and 64 scans. Quantitative analysis of cell macromolecular composition was done as in (Grube et al. 2002). As characteristic absorption bands were used 1080 cm−1 for carbohydrates, 1250 cm−1 for nucleic acids, 1550 cm−1 for proteins and 2930 cm−1 for lipids.Results and discussionsResults showed differences in biochemical composition of prostate and lung cancer cells even under normoxic growth conditions. The carbohydrate content in NCI-H69 cells was 8.16% of dry weight (dw), in PC3 cells – 11.60% dw. The lipid content in NCI-H69 cells was 10.91% dw, but in PC3%–13.58% dw. The hypoxia induced metabolic response of prostate and lung cancer cells was more pronounced as an increase of lipid content – e.g., the lipid content in hypoxic cells of NCI-H69 was 13.29% dw, but in PC3%–15.91% dw. Thus, hypoxia induced lipid increase in lung cancer cells was for 22% but in prostate cancer cells for 17% compared to that in normoxic cells. These results are in line with our previous results and thus might represent universal mechanism and the observed total lipid content increase due to hypoxia could have a potential value for further studies of cancer cell lipid metabolism.ConclusionThe results of this study showed that under oxygen stress – starvation (hypoxia) the metabolism of lipids was much more affected than that of proteins, nucleic acids or carbohydrates suggesting that regulation of lipid synthesis can possibly provide advantages for cancer cell survival in extreme growth conditions.

Effects of hypoxia on metabolic functions in marine organisms: Observed patterns and modelling assumptions within the context of Dynamic Energy Budget (DEB) theory

Hypoxia is a decrease in dissolved oxygen that causes physiological disturbances in marine fishes and invertebrates, including reduced mobility, growth rate and reproductive success, altered phenology and increased vulnerability to diseases. Under pressure from global changes such as warming or eutrophication, ocean and coastal ecosystems worldwide are becoming less oxygenated. In order to better understand the consequences of hypoxia on marine systems, there is a need for predicting hypoxia-induced changes from individual organisms to populations. By combining the effect of several stressors on species metabolic performances and life-history traits across their full life-cycle, the Dynamic Energy Budget (DEB) theory offers a suitable framework for studying the consequences of hypoxia on marine organisms and to envision an upscaling of these effects at population level through individual-based modelling approaches. Here, we propose a set of stylized biological facts which give a broad generalization of the effects of hypoxia on metabolic functions based on empirical findings. We used these stylized facts to derive assumptions on how to incorporate the effects of hypoxia on marine organisms in the framework of the DEB theory. We then validate some of these assumptions on marine species for which experimental datasets under hypoxic conditions and DEB parameters were available. We discuss the main issues that need to be dealt with, such as the various time-scales, ontogenic responses and multi-stressor effects which appear now as important targets of investigation for studying the effect of hypoxia on marine organisms.

Acute hypoxia alters reflex micturition behavior in urethane‐anesthetized carotid sinus intact and denervated adult female Sprague‐Dawley rat

Acute hypoxia is well known to increase respiratory drive by stimulating peripheral chemoreceptors located in the carotid bodies. Ongoing work in our laboratory has been examining the effects acute hypoxia on lower urinary tract (LUT) function in urethane‐anesthetized adult female rats. While these experiments have revealed that exposure to acute hypoxia is capable of modifying LUT function, the contribution of peripheral chemoreceptor‐mediated effects is unknown. Moreover, hypoxia‐induced modulation of LUT behavior is present both during hypoxia and transiently after the hypoxic stimulus is removed, such that hypoxia increases void frequency while the return to normoxia increases the threshold bladder pressure for voiding and prolongs the inter‐contraction interval (ICI). The present study was designed to evaluate whether the changes in LUT function noted are mediated by mechanisms that are dependent on carotid body hypoxia chemoreception. To this end, we conducted a series of experiments using continuous flow bladder cystometry with simultaneous recording of external urethral sphincter (EUS) EMG activity in urethane‐anesthetized spontaneously breathing adult female Sprague‐Dawley rats (n=10). Micturition events were elicited by saline infusion into the bladder via suprapubic catheter with the saline flow rate adjusted to induce a voiding frequency of approximately one void every 4–5 minutes. We also simultaneously recorded tongue, external oblique, and diaphragm muscle EMG activity via bipolar stainless steel EMG electrodes. Each rat was allowed to settle into a stable baseline voiding rhythm which was then recorded for approximately 1‐hr under normoxic conditions, after which an hypoxic challenge (12% O2, 15 min) was performed. In one group of rats, the carotid sinus nerves (CSN) were transected bilaterally prior to beginning the cytometry experiment while in a second group of rats the CSN were left intact. Success of the CSN denervation was confirmed by the absence of an increase in diaphragm EMG burst amplitude and frequency during the hypoxic challenge. Consistent with our prior observations, CSN intact rats responded to hypoxia with an increased voiding frequency, particularly at the onset of hypoxia; however, peak bladder pressure during contraction was variable with no consistent modulation by hypoxia. Upon return to normoxia, the initial voiding event was delayed, and subsequent events displayed increased ICI with elevated bladder pressure threshold. In contrast to CSN intact rats, CSN denervated rats exhibited a marked decrease in peak bladder contraction pressure during hypoxic exposure as well as a smaller increase in bladder pressure threshold after return to normoxia. No notable differences in hypoxia‐induced changes in ICI between CSN intact or transected rats was observed. These results suggest that hypoxia‐mediated carotid body afferent input is not required for producing a modulation of reflex micturition in response to hypoxia, but certain aspects of the hypoxic response may be impacted by carotid body dependent mechanisms.Support or Funding InformationNIH NS096514This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Ubiquitination of Profilin‐1 in Hypoxia‐Induced Pulmonary Hypertension

Pulmonary hypertension (PH) is a chronic illness characterized by increased pressure in the pulmonary arteries due to the thickening and constriction of the arterial walls. Vascular thickening is in part due to proliferation of smooth muscle cells. The ubiquitin proteasome system is an important regulator of cellular proliferation that alters protein stability and expression by modifying lysines (K) within target proteins with ubiquitin moieties. Addition of ubiquitin can alter activity of a protein or mark them for degradation. We hypothesized that hypoxia‐induced changes in ubiquitination impact PH development. Mass spectrometry (MS) was used to screen changes in ubiquitination of proteins in lung tissue from mice exposed to chronic hypoxia (10% O2) for 3 weeks. Profilin‐1, a mediator of actin‐polymerization, had decreased ubiquitination on K54 (Fold change −1.86) and K126 (Fold change −1.40). Profilin‐1 is known to play a role in cellular proliferation and its overexpression leads to increased wall thickness in aortas of spontaneously hypertensive rats. However, its role in PH is not well characterized. Proflin‐1 promotes actin polymerization by binding directly to ADP‐actin and catalyzing nucleotide exchange. Actin polymerization plays a key role in cellular proliferation, cell motility, and muscle contraction. We observed no difference in profilin‐1 protein levels in hypoxia‐exposed human pulmonary artery smooth muscle cells (HPASMC) and lungs from chronically hypoxia‐exposed mice when compared to normoxic controls (P&gt;0.05). This lack of an effect of ubiquitination on protein levels suggests that Profilin‐1 ubiquitination could regulate its activity rather than stability. Preliminary fractionate on results show increased association of Profilin‐1 and G‐actin in hypoxia. Furthermore, disrupting actin polymerization using actin polymerization inhibitors decreased HPASMC proliferation. Therefore, our results indicate that reduced levels of ubiquitinated Profilin‐1 may promote actin polymerization which increases proliferation and contributes to PH. In addition, they suggest that changes in protein ubiquitination may contribute to PH progression.Support or Funding InformationThis work was supported in part by Merit Review funding from the Department of Veterans Affairs, Office of Research and Development (1I01BX001910 to CMH), by NIH Grant R01HL102167 (to CMH and RLS), and by support from NIH T32 HL076118 and American Heart Association 16POST30930007 (to BEW). MS work was performed by the Emory Integrated Proteomics Core.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.