Low Oxygen Tension Research Articles

Microorganism-enabled photosynthetic oxygeneration and ferroptosis induction reshape tumor microenvironment for augmented nanodynamic therapy

Nanodynamic therapy (NDT) based on reactive oxygen species (ROS) generation has been envisioned as a distinct modality for efficient cancer treatment. However, insufficient ROS generation and partial ROS consumption frequently limit the theraputic effect and outcome of NDT owing to the low oxygen (O2) tension and high glutathione (GSH) level in tumor microenvironment (TME). To circumvent these critical issues, we herein proposed and engineered the biodegradable GSH-depletion Mn(III)-riched manganese oxide nanospikes (MnOx NSs) with the photosynthetic bacterial cyanobacteria (Cyan) as a high-efficient and synergistic platform to reshape TME by simultaneously increasing oxygen content and decreasing GSH level. Specifically, under the trigger of acidity, MnOx NSs reacted with photosynthetic oxygen can generate toxic singlet oxygen (1O2). Moreover, MnOx NSs significantly reduced intracellular GSH, resulting in decreased GPX4 activity, which induced tumor cell non-apoptotic ferroptosis. Consequently, this combined strategy based on coadministration with Cyan and MnOx NSs demonstrated the superior antitumor efficacy via amplification of oxidative stress in 4T1 tumor-bearing mice for the synergetic oxygen-augmented nanodynamic/ferroptosis therapy. This work highlights a facile synergistic micro-/nano-system with the specific capability of reshaping TME to augment the sensitivity and therapeutic efficacy of NDT in solid hypoxic tumor therapy.

HepaRG cells adopt zonal-like drug-metabolizing phenotypes under physiologically relevant oxygen tensions and Wnt/β-catenin signaling.

The cellular microenvironment plays an important role in liver zonation, the spatial distribution of metabolic tasks amongst hepatocytes lining the sinusoid. Standard tissue culture practices provide an excess of oxygen and a lack of signaling molecules typically found in the liver. We hypothesized that incorporating physiologically relevant environments would promote post-differentiation patterning of hepatocytes and result in zonal-like characteristics. To test this hypothesis, we evaluated the transcriptional regulation and activity of drug-metabolizing enzymes in HepaRG cells exposed to three different oxygen tensions, in the presence or absence of Wnt/β-catenin signaling. The drug-metabolizing activity of cells exposed to representative periportal (11% O2) or perivenous (5% O2) oxygen tensions were significantly less than cells exposed to ambient oxygen. A comparison of cytochrome P450 (CYP) 1A2, 2D6, and 3A4 activity at PP and PV oxygen tensions showed significant increases at the lower oxygen tension. The activation of the Wnt/β-catenin pathway only modestly impacted CYP activity at PV oxygen tension, despite a significant increase in CYP expression under this condition. Our results suggest oxygen tension is the major contributor to zonal patterning in HepaRG cells, with the Wnt/β-catenin signaling pathway playing a lesser albeit important role. Our datasets also highlight the importance of including activity-based assays, as transcript data alone does not provide an accurate picture of metabolic competence. Significance Statement This work investigates the post-differentiation patterning of HepaRG cells cultured at physiologically relevant oxygen tensions, in the presence and absence of Wnt/β-catenin signaling. HepaRG cells exposed to periportal (11% O2) or perivenous (5% O2) oxygen tensions display zonation-like patterning of both cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes. These datasets also suggest that oxygen is a primary regulator of post-differentiation patterning, with Wnt/β-catenin having a lesser effect on activity but a significant effect on transcriptional regulation of these enzymes.

Iron-regulated assembly of the cytosolic iron–sulfur cluster biogenesis machinery

The cytosolic iron–sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron–sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

Bladder cancer cell lines adapt their aggressiveness profile to oxygen tension.

During the process of tumor growth, cancer cells will be subjected to intermittent hypoxia. This results from the delay in the development of the vascular network in relation to the proliferation of cancer cells. The hypoxic nature of a tumor has been demonstrated as a negative factor for patient survival. To evaluate the impact of hypoxia on the survival and migration properties of low and high-grade bladder cancer cell lines, two low-grade (MGHU-3 and SW-780) and two high-grade (SW-1710 and T24) bladder cancer cell lines were cultured in normoxic (20% O2) or hypoxic atmospheric conditions (2% O2). The response of bladder cancer cell lines to hypoxic atmospheric cell culture conditions was examined under several parameters, including epithelial-mesenchymal transition, doubling time and metabolic activities, thrombospondin-1 expression, whole Matrix Metallo-Proteinase activity, migration and resistance to oxidative stress. The low-grade cell line response to hypoxia was heterogeneous even if it tended to adopt a more aggressive profile. Hypoxia enhanced migration and pro-survival properties of MGHU-3 cells, whereas these features were reduced for the SW-780 cell line cultured under low oxygen tension. The responses of tested high-grade cell lines were more homogeneous and tended to adopt a less aggressive profile. Hypoxia drastically changed some of the bladder cancer cell line properties, for example matrix metalloproteinases expression for all cancer cells but also switch in glycolytic metabolism of low grade cancer cells. Overall, studying bladder cancer cells in hypoxic environments are relevant for the translation from in vitro findings to in vivo context.

The double anaerobic threshold in heart failure: MECKI score database overview

AimsIn heart failure (HF), anaerobic threshold (AT) may be indeterminable but its value held a relevant prognostic role. AT is evaluated joining three methods: V‐slope, ventilatory equivalent, and end‐tidal methods. The possible non‐concordance between the V‐slope (met AT) and the other two methods (vent AT) has been highlighted in healthy individuals and named double threshold (DT).Methods and resultsWe reanalysed 1075 cardiopulmonary exercise tests of HF patients recruited in the Metabolic Exercise test data combined with Cardiac and Kidney Indexes (MECKI) score database. We identified DT in 43% of cases. Met AT precedes vent AT being met–ventΔVO2 221 (interquartile range: 129–319) mL/min. Peak VO2, 1307 ± 485 vs. 1343 ± 446 mL/min (63 ± 17 vs. 63 ± 17 percentage of predicted), was similar between DT+ and DT− patients. Differently, DT+ showed a lower ventilatory vs. carbon dioxide production (VE/VCO2) slope (29.6 ± 6.1 vs. 31.0 ± 6.3), a lower peak exercise end‐tidal oxygen tension (PetO2) 115.3 (111.5–118.9) vs. 116.4 (112.4–120.2) mmHg, and a higher carbon dioxide tension (PetCO2) 34.2 (30.9–37.1) vs. 32.4 (28.7–35.5) mmHg. Vent AT showed a significant higher VO2, 957 ± 318 vs. 719 ± 252 mL/min, VCO2, 939 ± 319 vs. 627 ± 226 mL/min, ventilation, 31.0 ± 8.3 vs. 22.5 ± 6.3 L/min, respiratory exchange ratio, 0.98 ± 0.08 vs. 0.87 ± 0.07, PetO2, 108 (104–112) vs. 105 (101–109) mmHg, PetCO2, 37 (34–40) vs. 36 (33–39) mmHg, and VE/VO2 ratio, 33.5 ± 6.7 vs. 32.6 ± 6.9, but lower VE/VCO2 ratio, 33 (30–37) vs. 36 (32–41), compared with met AT. At 2 year survival by Kaplan–Meier analysis, even adjusted for confounders, DT resulted not associated with survival.ConclusionsDouble threshold is frequently observed in HF patients. DT+ is associated to a decreased ventilatory response during exercise.

Targeting Hypoxia‐Adenosinergic Signaling to Enable Effective Anti‐Tumor Responses by Reprogramming the Immunosuppressive Tumor Microenvironment

The hypoxia‐HIF‐1ɑ‐mediated generation of extracellular adenosine (eADO) and associated A2A adenosine receptor (A2AR) signaling represent a critical mechanism for resistance to anti‐tumor immune attack in an immunosuppressive tumor microenvironment (TME). This is supported by earlier clinical and preclinical findings that tumors exhibiting refractory responses to chemotherapeutic and immunotherapeutic interventions are often characterized by low tissue oxygen tension and elevation of intracellular cAMP triggered by the binding of adenosine to the A2AR. The ensuing signaling cascade results in the downregulation of T cell activation and increased production of anti‐inflammatory factors. Several classes of drugs have been proven in preclinical studies and clinical trials to weaken this powerful biochemical mechanism of immunosuppression and enable tumor rejection. Therapeutic approaches targeting the upstream and downstream stages of hypoxia‐adenosinergic negative immune regulation (i.e. A2AR antagonists, oxygenation, inhibition of adenosine generation or adenosine degradation) represent promising co‐adjuvants capable of inducing a more immunopermissive TME. The removal of this biochemical barrier may be the key to unleashing the full therapeutic potential of immunotherapies. For instance, the epithelial‐mesenchymal transition (EMT) program, which confers the ability of cancer cells to engage in metastatic dissemination, is associated with the acquisition of resistance to immune checkpoint blockade (ICB). Recent studies have demonstrated that abrogation of the adenosine‐generating ectoenzyme CD73 sensitizes mice bearing quasi‐mesenchymal (qM) tumors to anti‐CTLA4 ICB through an increase in CD8+ T cell infiltration and cytotoxicity. Furthermore, the present study demonstrates the differential expression of adenosinergic enzymes and EMT transcription factors (EMT‐TFs) in murine breast epithelial (E) and qM carcinoma cell lines cultured in normoxic (21% oxygen) and hypoxic (1% oxygen) conditions in vitro. As hypothesized, the stabilization of HIF‐1ɑ in hypoxic conditions resulted in the upregulation of some adenosine‐generating enzymes and changes in adenosine metabolism. These results elucidate an additional mechanism through which hypoxia can contribute to the assembly of an immunosuppressive TME, while highlighting the categorical differences between the E vs qM states of carcinoma cells.

Microfluidic Microcirculation Mimetic as a Tool for the Study of Rheological Characteristics of Red Blood Cells in Patients with Sickle Cell Anemia

Sickle cell disorder (SCD) is a multisystem disease with heterogeneous phenotypes. Although all patients have the mutated hemoglobin (Hb) in the SS phenotype, the severity and frequency of complications are variable. When exposed to low oxygen tension, the Hb molecule becomes dense and forms tactoids, which lead to the peculiar sickled shapes of the affected red blood cells, giving the disorder its name. This sickle cell morphology is responsible for the profound and widespread pathologies associated with this disorder, such as vaso-occlusive crisis (VOC). How much of the clinical manifestation is due to sickled erythrocytes and what is due to the relative contributions of other elements in the blood, especially in the microcapillary circulation, is usually not visualized and quantified for each patient during clinical management. Here, we used a microfluidic microcirculation mimetic (MMM), which has 187 capillary-like constrictions, to impose deformations on erythrocytes of 25 SCD patients, visualizing and characterizing the morpho-rheological properties of the cells in normoxic, hypoxic (using sodium meta-bisulfite) and treatment conditions (using hydroxyurea). The MMM enabled a patient-specific quantification of shape descriptors (circularity and roundness) and transit time through the capillary constrictions, which are readouts for morpho-rheological properties implicated in VOC. Transit times varied significantly (p < 0.001) between patients. Our results demonstrate the feasibility of microfluidics-based monitoring of individual patients for personalized care in the context of SCD complications such as VOC, even in resource-constrained settings.

Engineering Vascularized Islet Macroencapsulation Devices: An in vitro Platform to Study Oxygen Transport in Perfused Immobilized Pancreatic Beta Cell Cultures.

Islet encapsulation devices serve to deliver pancreatic beta cells to type 1 diabetic patients without the need for chronic immunosuppression. However, clinical translation is hampered by mass transport limitations causing graft hypoxia. This is exacerbated in devices relying only on passive diffusion for oxygenation. Here, we describe the application of a cylindrical in vitro perfusion system to study oxygen effects on islet-like clusters immobilized in alginate hydrogel. Mouse insulinoma 6 islet-like clusters were generated using microwell plates and characterized with respect to size distribution, viability, and oxygen consumption rate to determine an appropriate seeding density for perfusion studies. Immobilized clusters were perfused through a central channel at different oxygen tensions. Analysis of histological staining indicated the distribution of viable clusters was severely limited to near the perfusion channel at low oxygen tensions, while the distribution was broadest at normoxia. The results agreed with a 3D computational model designed to simulate the oxygen distribution within the perfusion device. Further simulations were generated to predict device performance with human islets under in vitro and in vivo conditions. The combination of experimental and computational findings suggest that a multichannel perfusion strategy could support in vivo viability and function of a therapeutic islet dose.

HIF-1-Dependent Induction of β3 Adrenoceptor: Evidence from the Mouse Retina.

A major player in the homeostatic response to hypoxia is the hypoxia-inducible factor (HIF)-1 that transactivates a number of genes involved in neovessel proliferation in response to low oxygen tension. In the retina, hypoxia overstimulates β-adrenoceptors (β-ARs) which play a key role in the formation of pathogenic blood vessels. Among β-ARs, β3-AR expression is increased in proliferating vessels in concomitance with increased levels of HIF-1α and vascular endothelial growth factor (VEGF). Whether, similarly to VEGF, hypoxia-induced β3-AR upregulation is driven by HIF-1 is still unknown. We used the mouse model of oxygen-induced retinopathy (OIR), an acknowledged model of retinal angiogenesis, to verify the hypothesis of β3-AR transcriptional regulation by HIF-1. Investigation of β3-AR regulation over OIR progression revealed that the expression profile of β3-AR depends on oxygen tension, similar to VEGF. The additional evidence that HIF-1α stabilization decouples β3-AR expression from oxygen levels further indicates that HIF-1 regulates the expression of the β3-AR gene in the retina. Bioinformatics predicted the presence of six HIF-1 binding sites (HBS #1-6) upstream and inside the mouse β3-AR gene. Among these, HBS #1 has been identified as the most suitable HBS for HIF-1 binding. Chromatin immunoprecipitation-qPCR demonstrated an effective binding of HIF-1 to HBS #1 indicating the existence of a physical interaction between HIF-1 and the β3-AR gene. The additional finding that β3-AR gene expression is concomitantly activated indicates the possibility that HIF-1 transactivates the β3-AR gene. Our results are indicative of β3-AR involvement in HIF-1-mediated response to hypoxia.

Pulmonary and laryngeal oedema before induction of anaesthesia in a sheep

AbstractThis case report describes the anaesthesia of a ram undergoing an exploratory laparotomy, which suffered from respiratory distress during the perioperative period. On the morning of surgery, the animal presented with oedematous ears, most likely caused by a tight‐fitting muzzle that had been used for preoperative fasting. After sedation, a mild stridor was heard. During endotracheal intubation, a swollen larynx was observed, and a frothy discharge emerged through the endotracheal tube after placement. Most likely, the tight muzzle caused a partial obstruction of the nostrils, increasing the respiratory effort and causing an increase in the pressure gradient, which provoked laryngeal and pulmonary oedema. Treatment consisted of repeated suctioning of fluid from the endotracheal tube, furosemide, dexamethasone, mechanical ventilation with 100% oxygen during anaesthesia and oxygen supplementation during recovery. Despite moderately low arterial oxygen tension during anaesthesia, the animal recovered uneventfully and did not show further signs of respiratory distress.

Hypoxia Induced Changes of Exosome Cargo and Subsequent Biological Effects.

Exosomes are small extracellular vesicles that are secreted by almost all types of cells and exist in almost all extracellular spaces. As an important mediator of intercellular communication, exosomes encapsulate the miRNA, lncRNA, cirRNA, mRNA, cytokine, enzyme, lipid, and other components from the cytoplasm into its closed single membrane structure and transfer them to recipient units in an autocrine, paracrine, or endocrine manner. Hypoxia is a state of low oxygen tension and is involved in many pathological processes. Hypoxia influences the size, quantity, and expression of exosome cargos. Exosomes derived from hypoxic tumor cells transfer genetics, proteins, and lipids to the recipient units to exert pleiotropic effects. Different donor cells produce different cargo contents, target different recipient units and lead to different biological effects. Hypoxic exosomes derived from tumor cells uptaken by normoxic tumor cells lead to promoted proliferation, migration, and invasion; uptaken by extracellular space or liver lead to promoted metastasis; uptaken by endothelial cells lead to promoted angiogenesis; uptaken by immune cells lead to promoted macrophage polarization and changed tumor immune microenvironment. In addition to various types of tumors, hypoxic exosomes also participate in the development of diseases in the cardiovascular system, neuron system, respiratory system, hematology system, endocrine system, urinary system, reproduction system, and skeletomuscular system. Understanding the special characteristics of hypoxic exosomes provide new insight into elaborating the pathogenesis of hypoxia related disease. This review summarizes hypoxia induced cargo changes and the biological effects of hypoxic exosomes in tumors and non-malignant diseases in different systems.

Hypoxia preconditioning elicit differential response in tissue-specific MSCs via immunomodulation and exosomal secretion.

Mesenchymal stromal cells (MSCs) are emerging as an ideal candidate for regenerative medicine. It is known that the culture conditions impact the cellular properties of MSCs and their therapeutic behavior. Moreover, maintenance of MSCs in low oxygen tension for a short duration has shown to be beneficial for MSCs as it is similar to that of their physiological niche. However, the precise mechanism through which hypoxia pre-conditioning affects MSCs is not clear yet. Thus, in this study, we have investigated the effect of hypoxia exposure (1% O2) on tissue-specific MSCs over a period of time under serum-free culture conditions and evaluated the changes in expression of immuno-modulatory molecules and exosome biogenesis and secretion markers. It was observed that all MSCs responded differentially towards hypoxia exposure as indicated by the expression of HIF-1α. Moreover, this short-term exposure did not induce any changes in MSCs cellular morphology, proliferation rate, and surface marker profiling. In addition, we observed an enhancement in the expression of immunomodulatory factors (HLA-G, PGE-2, and IDO) after hypoxia exposure of 12 to 24h in all tissue-specific MSCs. Interestingly, we have also observed the upregulation in exosome secretion that was further corelated to the upregulation of expression of exosome biogenesis and secretion markers (ALIX, TSG101, RAB27a, RAB27b). Though there was a differential response of MSCs where WJ-MSCs and BM-MSCs showed upregulation of these markers at 6-12h of hypoxia pre-conditioning, while AD-MSCs showed similar changes beyond 24h of hypoxia exposure.

Metabolic Determinants of Cardiomyocyte Proliferation.

The adult mammalian heart is recalcitrant to regeneration after injury, in part due to the postmitotic nature of cardiomyocytes. Accumulating evidence suggests that cardiomyocyte proliferation in fetal or neonatal mammals and in regenerative non-mammalian models depends on a conducive metabolic state. Results from numerous studies in adult hearts indicate that conditions of relatively low fatty acid oxidation, low reactive oxygen species generation, and high glycolysis are required for induction of cardiomyocyte proliferation. Glycolysis appears particularly important because it provides branchpoint metabolites for several biosynthetic pathways that are essential for synthesis of nucleotides and nucleotide sugars, amino acids, and glycerophospholipids, all of which are required for daughter cell formation. In addition, the proliferative cardiomyocyte phenotype is supported in part by relatively low oxygen tensions and through the actions of critical transcription factors, coactivators, and signaling pathways that promote a more glycolytic and proliferative cardiomyocyte phenotype, such as hypoxia inducible factor 1α (Hif1α), Yes-associated protein (Yap), and ErbB2. Interventions that inhibit glycolysis or its integrated biosynthetic pathways almost universally impair cardiomyocyte proliferative capacity. Furthermore, metabolic enzymes that augment biosynthetic capacity such as phosphoenolpyruvate carboxykinase 2 and pyruvate kinase M2 appear to be amplifiers of cardiomyocyte proliferation. Collectively, these studies suggest that acquisition of a glycolytic and biosynthetic metabolic phenotype is a sine qua non of cardiomyocyte proliferation. Further knowledge of the regulatory mechanisms that control substrate partitioning to coordinate biosynthesis with energy provision could be leveraged to prompt or augment cardiomyocyte division and to promote cardiac repair.

An observational study of simultaneous pulse oximetry and arterial oxygen saturation readings in intensive care unit/high dependency unit in COVID-19 patients

Background: The coronavirus-2019 (COVID-19) pandemic has increased the use of pulse oximeters worldwide. It has become an inevitable tool in the monitoring of the disease. However, the accuracy of pulse oximeters in COVID-19 has not been established. Aims and Objectives: The aims of the study were to examine the relationship between oxygen saturation as measured by pulse oximeter (SpO2) and oxygen saturation measured by arterial blood gas analysis (SaO2) measurements in COVID-19 patients admitted to the intensive care unit (ICU)/high dependency unit (HDU) and to assess the ability of SpO2 readings to detect low SaO2 and low oxygen tension in COVID-19 patients. Materials and Methods: This prospective observational study was conducted in the COVID-19 ICU and high dependency unit of a tertiary care hospital in Bengaluru, India. All patients admitted with confirmed COVID-19, meeting the eligibility criteria, were included in the study. We assessed bias and limits of agreement between paired samples of oxygen saturation from pulse oximetry (SpO2) and arterial oxygen saturation from blood gas analysis (SaO2). Results: The sample mean difference SpO2-SaO2 is −0.86% (bias) and the 95% confidence interval for the mean difference was −1.67 and −0.04. The lower limit of agreement was −7.32 with a 95% confidence interval (−8.74, −5.91). The upper limit of agreement was 5.61 with a 95% confidence interval of 4.19 and 7.02. Conclusion: SpO2 values are not completely dependable in estimating SaO2 in COVID-19 patients in ICU/HDU; therefore, arterial blood gas analysis measurement of oxygen saturation has to be done depending on the clinical scenario CTRI (CTRI/2020/11/029035).

Hypoxia Promotes Neutrophil Survival After Acute Myocardial Infarction.

Phagocytosis, degranulation, and neutrophil extracellular traps (NETs) formation build the armory of neutrophils for the first line of defense against invading pathogens. All these processes are modulated by the microenvironment including tonicity, pH and oxygen levels. Here we investigated the neutrophil infiltration in cardiac tissue autopsy samples of patients with acute myocardial infarction (AMI) and compared these with tissues from patients with sepsis, endocarditis, dermal inflammation, abscesses and diseases with prominent neutrophil infiltration. We observed many neutrophils infiltrating the heart muscle after myocardial infarction. Most of these had viable morphology and only few showed signs of nuclear de-condensation, a hallmark of early NET formation. The abundance of NETs was the lowest in acute myocardial infarction when compared to other examined diseases. Since cardiac oxygen supply is abruptly abrogated in acute myocardial infarction, we hypothesized that the resulting tissue hypoxia increased the longevity of the neutrophils. Indeed, the viable cells showed increased nuclear hypoxia inducible factor-1α (HIF-1α) content, and only neutrophils with low HIF-1α started the process of NET formation (chromatin de-condensation and nuclear swelling). Prolonged neutrophil survival, increased oxidative burst and reduced NETs formation were reproduced under low oxygen tensions and by HIF-1α stabilization in vitro. We conclude that nuclear HIF-1α is associated with prolonged neutrophil survival and enhanced oxidative stress in hypoxic areas of AMI.

The effect of hypoxia on myogenic differentiation and multipotency of the skeletal muscle-derived stem cells in mice

BackgroundSkeletal muscle-derived stem cells (SC) have become a promising approach for investigating myogenic differentiation and optimizing tissue regeneration. Muscle regeneration is performed by SC, a self-renewal cell population underlying the basal lamina of muscle fibers. Here, we examined the impact of hypoxia condition on the regenerative capacity of SC either in their native microenvironment or via isolation in a monolayer culture using ectopic differentiation inductions. Furthermore, the effect of low oxygen tension on myogenic differentiation protocols of the myoblasts cell line C2C12 was examined.MethodsHind limb muscles of wild type mice were processed for both SC/fiber isolation and myoblast extraction using magnetic beads. SC were induced for myogenic, adipogenic and osteogenic commitments under normoxic (21% O2) and hypoxic (3% O2) conditions. SC proliferation and differentiation were evaluated using histological staining, immunohistochemistry, morphometric analysis and RT-qPCR. The data were statistically analyzed using ANOVA.ResultsThe data revealed enhanced SC proliferation and motility following differentiation induction after 48 h under hypoxia. Following myogenic induction, the number of undifferentiated cells positive for Pax7 were increased at 72 h under hypoxia. Hypoxia upregulated MyoD and downregulated Myogenin expression at day-7 post-myogenic induction. Hypoxia promoted both SC adipogenesis and osteogenesis under respective induction as shown by using Oil Red O and Alizarin Red S staining. The expression of adipogenic markers; peroxisome proliferator activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4) were upregulated under hypoxia up to day 14 compared to normoxic condition. Enhanced osteogenic differentiation was detected under hypoxic condition via upregulation of osteocalcin and osteopontin expression up to day 14 as well as, increased calcium deposition at day 21. Hypoxia exposure increases the number of adipocytes and the size of fat vacuoles per adipocyte compared to normoxic culture. Combining the differentiation medium with dexamethasone under hypoxia improves the efficiency of the myogenic differentiation protocol of C2C12 by increasing the length of the myotubes.ConclusionsHypoxia exposure increases cell resources for clinical applications and promotes SC multipotency and thus beneficial for tissue regeneration.

Hypoxia-Conditioned Mesenchymal Stem Cells in Tissue Regeneration Application.

Mesenchymal stem cells (MSCs) have been demonstrated as promising cell sources for tissue regeneration due to their capability of self-regeneration, differentiation, and immunomodulation. MSCs also exert extensive paracrine effects through release of trophic factors and extracellular vesicles (EVs). However, despite extended exploration of MSCs in preclinical studies, the results are far from satisfactory due to the poor engraftment and low level of survival after implantation. Hypoxia preconditioning has been proposed as an engineering approach to improve the therapeutic potential of MSCs. During in vitro culture, hypoxic conditions can promote MSC proliferation, survival, and migration through various cellular responses to the reduction of oxygen tension. The multilineage differentiation potential of MSCs is altered under hypoxia, with consistent reports of enhanced chondrogenesis. Hypoxia also stimulates the paracrine activities of MSCs and increases the production of secretome both in terms of soluble factors as well as EVs. The secretome from hypoxia-preconditioned MSCs play important roles in promoting cell proliferation and migration, enhancing angiogenesis while inhibiting apoptosis and inflammation. In this review, we summarize current knowledge of hypoxia-induced changes in MSCs and discuss the application of hypoxia-preconditioned MSCs as well as hypoxic secretome in different kinds of disease models. Impact statement Mesenchymal stem cells (MSCs) have been applied in numerous cell-based and secretome-based therapies for tissue regeneration. Hypoxic conditions enhance the function of MSCs by increasing proliferation, survival, homing, differentiation, and paracrine activities. A timely up-to-date comprehensive overview of the effect of low oxygen tension to MSC, with emphasis on the influence and molecular mechanism of hypoxia preconditioning toward MSC's functionality is provided, including the therapeutic use of hypoxia-preconditioned MSC as well as hypoxic secretome in various prove-of-concept disease models. This knowledge would contribute to future engineering of MSC culture conditions for improved translational application.

Molecular Characterization and Response of Prolyl Hydroxylase Domain (PHD) Genes to Hypoxia Stress in Hypophthalmichthys molitrix

Simple SummaryHypoxia is a common challenge for aquatic organisms, and prolyl hydroxylase domain (PHD) proteins play important roles in hypoxic adaptation by regulating the stability of the hypoxia-inducible factor 1 alpha subunit (HIF-1α). In this study, the full-length cDNAs of three PHD genes were obtained from Hypophthalmichthys molitrix, which is an important freshwater fish and sensitive to low oxygen tension. The amino acid sequence analysis and phylogenetic analysis of PHDs were performed among various species. Furthermore, the expression patterns and the transcriptional responses of H. molitrix PHD genes to acute hypoxia, continued hypoxia, and reoxygenation were explored in different tissues. Our study preliminarily explored the physiological regulation functions of PHD genes at the transcriptional level when addressing the hypoxic challenge and provided a foundation for future systematic explorations of the molecular mechanisms underlying hypoxia adaptation in silver carp.As an economically and ecologically important freshwater fish, silver carp (Hypophthalmichthys molitrix) is sensitive to low oxygen tension. Prolyl hydroxylase domain (PHD) proteins are critical regulators of adaptive responses to hypoxia for their function of regulating the hypoxia inducible factor-1 alpha subunit (HIF-1α) stability via hydroxylation reaction. In the present study, three PHD genes were cloned from H. molitrix by rapid amplification of cDNA ends (RACE). The total length of HmPHD1, HmPHD2, and HmPHD3 were 2981, 1954, and 1847 base pair (bp), and contained 1449, 1080, and 738 bp open reading frames (ORFs) that encoded 482, 359, and 245 amino acids (aa), respectively. Amino acid sequence analysis showed that HmPHD1, HmPHD2, and HmPHD3 had the conserved prolyl 4-hydroxylase alpha subunit homolog domains at their C-termini. Meanwhile, the evaluation of phylogeny revealed PHD2 and PHD3 of H. molitrix were more closely related as they belonged to sister clades, whereas the clade of PHD1 was relatively distant from these two. The transcripts of PHD genes are ubiquitously distributed in H. molitrix tissues, with the highest expressional level of HmPHD1 and HmPHD3 in liver, and HmPHD2 in muscle. After acute hypoxic treatment for 0.5 h, PHD genes of H. molitrix were induced mainly in liver and brain, and different from HmPHD1 and HmPHD2, the expression of HmPHD3 showed no overt tissue specificity. Furthermore, under continued hypoxic condition, PHD genes exhibited an obviously rapid but gradually attenuated response from 3 h to 24 h, and upon reoxygenation, the transcriptional expression of PHD genes showed a decreasing trend in most of the tissues. These results indicate that the PHD genes of H. molitrix are involved in the early response to hypoxic stress, and they show tissue-specific transcript expression when performing physiological regulation functions. This study is of great relevance for advancing our understanding of how PHD genes are regulated when addressing the hypoxic challenge and provides a reference for the subsequent research of the molecular mechanisms underlying hypoxia adaptation in silver carp.

Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion.

Rhomboids are intramembrane serine proteases highly conserved in the three domains of life. Their key roles in eukaryotes are well understood but their contribution to bacterial physiology is still poorly characterized. Here we demonstrate that Brucella abortus, the etiological agent of the zoonosis called brucellosis, encodes an active rhomboid protease capable of cleaving model heterologous substrates like Drosophila melanogaster Gurken and Providencia stuartii TatA. To address the impact of rhomboid deletion on B. abortus physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. About 50% of the B. abortus predicted proteome was identified by quantitative proteomics under two experimental conditions and 108 differentially represented proteins were detected. Membrane associated proteins that showed variations in concentration in the mutant were considered as potential rhomboid targets. This class included nitric oxide reductase subunit C NorC (Q2YJT6) and periplasmic protein LptC involved in LPS transport to the outer membrane (Q2YP16). Differences in secretory proteins were also addressed. Differentially represented proteins included a putative lytic murein transglycosylase (Q2YIT4), nitrous-oxide reductase NosZ (Q2YJW2) and high oxygen affinity Cbb3-type cytochrome c oxidase subunit (Q2YM85). Deletion of rhomboid had no obvious effect in B. abortus virulence. However, rhomboid overexpression had a negative impact on growth under static conditions, suggesting an effect on denitrification enzymes and/or high oxygen affinity cytochrome c oxidase required for growth in low oxygen tension conditions.

Effects of oxygen tension and humidity on the preimplantation development of mouse embryos produced by in vitro fertilization: analysis using a non-humidifying incubator with time-lapse cinematography.

To examine the effects of oxygen tension and humidity on early embryonic development, the preimplantation development of mouse embryos produced by in vitro fertilization was assessed by time-lapse cinematography to evaluate morphokinetic development with higher precision. Zygotes were produced from spermatozoa and oocytes from ICR mice and cultured in KSOM under low or high oxygen tension in a non-humidified incubator with time-lapse cinematography (CCM-iBIS). The developmental rates of embryos to the 4-cell and blastocyst stages under lower oxygen tension in CCM-iBIS were significantly higher than those under higher oxygen tension in CCM-iBIS. Ninety-six hours after insemination, a large number of embryos cultured under low oxygen tension developed to the hatching blastocyst stage. Embryonic development was more synchronized under lower oxygen tension. Non-humidified cultures did not affect embryonic development. On average, mouse embryos cultured at lower oxygen tension reached 2-cell at 18 h, 3-cell at 39 h, 4-cell at 40 h, initiation of compaction at 58 h, morula at 69 h, and blastocyst at 82 h after insemination. In conclusion, lower oxygen tension better supports preimplantation development of mouse embryos fertilized in vitro, and non-humidified culture conditions do not influence the embryonic development in vitro.