Normoxic Environment Research Articles

Flavylium-Based Hypoxia-Responsive Probe for Cancer Cell Imaging.

A hypoxia-responsive probe based on a flavylium dye containing an azo group (AZO-Flav) was synthesized to detect hypoxic conditions via a reductase-catalyzed reaction in cancer cells. In in vitro enzymatic investigation, the azo group of AZO-Flav was reduced by a reductase in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) followed by fragmentation to generate a fluorescent molecule, Flav-NH2. The response of AZO-Flav to the reductase was as fast as 2 min with a limit of detection (LOD) of 0.4 μM. Moreover, AZO-Flav displayed high enzyme specificity even in the presence of high concentrations of biological interferences, such as reducing agents and biothiols. Therefore, AZO-Flav was tested to detect hypoxic and normoxic environments in cancer cells (HepG2). Compared to the normal condition, the fluorescence intensity in hypoxic conditions increased about 10-fold after 15 min. Prolonged incubation showed a 26-fold higher fluorescent intensity after 60 min. In addition, the fluorescence signal under hypoxia can be suppressed by an electron transport process inhibitor, diphenyliodonium chloride (DPIC), suggesting that reductases take part in the azo group reduction of AZO-Flav in a hypoxic environment. Therefore, this probe showed great potential application toward in vivo hypoxia detection.

Effect of pulsatile stretch on unfolded protein response in a new model of the pulmonary hypertensive vascular wall

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by hypoxemia and arterial remodeling. Dynamic stretch and recoil of the arterial wall during pulsation (in normal conduit arteries, stretch 20% above diastolic diameter) maintains homeostasis; a static arterial wall is associated with remodeling. PPHN is diagnosed by echocardiography as decreased pulmonary artery wall displacement during systole, causing decreased pulmonary arterial pressure acceleration time in a stiff artery.We hypothesized that a ‘normal’ amplitude of pulsatile stretch is protective against ER stress, while the loss of stretch is a trigger for hypoxia-induced stress responses. Using a novel in vitro model of pulmonary arterial myocytes subject to repetitive stretch-relaxation cycles within a normoxic or hypoxic environment, we examined the relative impact of hypoxia (pulmonary circuit during unresolved PPHN) and cyclic mechanical stretch (diminished in PPHN) on myocyte homeostasis, specifically on signaling proteins for autophagy and endoplasmic reticulum (ER) stress.Stretch induced autophagosome abundance under electron microscopy. Hypoxia, in presence or absence of pulsatile stretch, decreased unfolded protein response (UPR) hallmark BIP (GRP78) in contractile phenotype pulmonary arterial myocytes. Inositol requiring enzyme-1 α (IRE1α) was not activated; but hypoxia induced eif2α phosphorylation, increasing expression of ATF4 (activating transcription factor-4). This was sensitive to inhibition by autophagy inhibitor bafilomycin A1.We conclude that in the pulmonary circuit, hypoxia induces one arm of the UPR pathway and causes ER stress. Pulsatile stretch ameliorates the hypoxic UPR response, and while increasing presence of autophagosomes, does not activate canonical autophagy signaling pathways. We propose that simultaneous application of hypoxia and graded levels of cyclic stretch can be used to distinguish myocyte signaling in the deformable pulmonary artery of early PPHN, versus the inflexible late stage PPHN artery.

Noval Insights into the Role of Platelet Angiogenic Growth Factors on the Regulation of Normal Vascular Development

The role of platelets as angiogenic regulators has been the subject of intense research over the last decade, and has led to the recognition of platelets as the predominant source of key angiogenic growth factors (AGFs) in the circulation. Through the regulated delivery of these factors, platelets contribute to wound healing, maintenance of vascular integrity, and tumor angiogenesis. Prior studies have shown that thrombocytopenia in adult mice leads to hemorrhages primarily from structurally abnormal new blood vessels, both in in vivo angiogenesis assays and inside tumors. The rapid growth during fetal and neonatal life is associated with a developmental stage-specific need for accelerated angiogenesis and vascular development. However, whether and how platelets participate in normal fetal/neonatal vascular development is unknown, except for their role in blood/lymphatic separation. We hypothesized that platelets would be important for normal fetal/neonatal vascular development, and that their AGF profile would be different from that of adult platelets. As a first step, we quantified five key AGFs (VEGF-A, bFGF, ANG-1, PDGF-BB and TGF-b) by ELISA in platelet lysates and platelet-poor plasma from term cord blood samples (CB; n=12) and from the peripheral blood of healthy adult volunteers (PB; n=8). These studies demonstrated that CB platelets, just like PB platelets, are the major source of AGFs in the circulation, carrying more than 80% of the levels of these AGFs present in the blood. However, the angiogenic profiles were different in platelets of different developmental origins. Fetal/neonatal platelets contained ~70% more bFGF and VEGF-A than adult platelets (p<0.01 for both), but had significantly lower levels of ANG-1, PDGF-BB and TGF-β1 (p<0.05 vs. adult platelets). VEGF-A and bFGF are essential for the proliferation and differentiation of endothelial cells, while ANG-1, PDGFs and TGF-b concert mural cell coverage, basement membrane formation and vascular maturation, thus stabilizing and “strengthening” the vasculature. Based on these characteristics, we hypothesized that neonatal and adult platelets would have different biological effects. To test this, we first assessed the effects of CB- vs. PB-derived platelet lysates on human umbilical vascular endothelial cells (HUVEC), in a tube formation assay. In these experiments, CB platelets more potently stimulated angiogenesis, quantified as number of capillary branch points at 4, 5, and 6 hours, than PB platelets. Subsequently, we compared the proliferative effects of CB vs. PB platelet lysates on human primary CB-derived endothelial progenitor cells (EPCs) and amniotic membrane-derived mesenchymal stromal cells (MSCs). Consistent with the effects on HUVEC cells, CB platelet lysates induced approximately 50% more proliferation of EPCs, compared to PB platelet lysates (p<0.001). In contrast, PB platelet lysates were approximately twice more potent in promoting MSCs growth (p<0.001 vs. CB platelets). Since EPCs give rise to the vascular endothelium and MSCs differentiate into mural cells, these results strongly suggested that the AGF content in fetal/neonatal platelets promotes rapid angiogenesis at the expense of a stable vasculature, matching the developmental needs of rapidly growing fetuses and neonates. Finally, since birth is associated with an abrupt change in oxygen concentration (from the profoundly hypoxic intrauterine to the normoxic extrauterine environment), we assessed whether oxygen concentration would modulate the VEGF content in CB-derived cultured MKs (and thus platelets). Consistent with this hypothesis, CB MKs cultured in 5% oxygen released significantly more VEGF in the medium compared to MKs cultured in 20% oxygen (p<0.05). Our findings strongly suggest that the platelet AGF profile changes from neonatal to adult life to help regulate vascular development at different stages of life, and suggest that it might also change in response to environmental factors such as oxygen concentration. Whether a change in platelet angiogenic profile associated with the sudden increase in oxygen concentration during preterm birth contributes to diseases of prematurity characterized by disrupted vascular development (i.e. retinopathy of prematurity) requires further investigation. DisclosuresSola-Visner:Sysmex Inc.: Research Funding.

Mitochondria-targeted nanotheranostic: Harnessing single-laser-activated dual phototherapeutic processing for hypoxic tumor treatment

<h2>Summary</h2> Realizing maximum tumor suppression along with preventing tumor regrowth by optimizing the photon usage in phototherapy remains a major challenge. Herein, a mitochondria-targeted phototheranostic nanoformulation (<b>MsPDTT NPs</b>) was prepared from a molecular theranostic encapsulated into phospholipids. Notably, under single 690 nm laser excitation, <b>MsPDTT NPs</b> can perform dual-mode photoacoustic and near-infrared fluorescence imaging and potent photodynamic therapy/photothermal therapy (PDT/PTT) because of efficient excited-state deactivation pathways (through radiative and energy transfer to generate reactive oxygen species and non-radiative relaxation). The reference <b>RsPDTT NPs</b> lacking the mitochondria-targeting feature exhibit only the PTT property. Based on biological results, the <b>MsPDTT NP</b> therapeutic response can be switched to PDT and PTT under normoxic and hypoxic environments and maximize the overall efficacy of phototherapies without any noticeable side effects. The current findings suggest the potential of using simultaneous PDT/PTT with proper photon utilization as a promising theranostic approach for hypoxic tumor photoablation.

Effects of in vitro low oxygen tension preconditioning of buccal fat pad stem cells on in Vivo articular cartilage tissue repair

AimsProgenitor cells-based regenerative strategy has shown promise to repair cartilage, an avascular tissue in which cells experience hypoxia. Hypoxia is known to improve the early chondrogenic differentiation of stem cells. Therefore, this study aimed to determine whether hypoxia preconditioning could be used to enhance the regenerative potential of the combination of buccal fat pad stem cells (BFPSCs) and bilayer chitosan-based hydrogel scaffold for articular cartilage repair. Materials and methodsHuman BFPSCs were seeded on the bilayer chitosan-based hydrogel scaffolds in the culture medium. The viability and proliferation of cells on the scaffolds were monitored using scanning electron microscopy (SEM), MTT assay, and DAPI staining. Hypoxia preconditioned BFPSCs-seeded scaffolds were transplanted into rabbit articular cartilage knee defects for 12 weeks. The newly formed tissue was evaluated by cartilage-specific immunohistological analysis and histological staining. Key findingsIt was found that the chondrogenic differentiation and osteochondral conjunction in articular cartilage defect via BFPSCs-seeded bilayer scaffolds was enhanced by hypoxic preconditioning compared to a normoxic environment. SignificanceBased on our study, the integrity with subchondral bone in osteochondral defect was enhanced by BFPSCs on bilayer scaffold. Thus, this study provides evidence on the design of preconditioned cell-seeded bilayer hydrogels for articular cartilage regeneration.

Genome-wide association analysis of adaptation to oxygen stress in Nile tilapia (Oreochromis niloticus)

BackgroundTilapia is one of the most abundant species in aquaculture. Hypoxia is known to depress growth rate, but the genetic mechanism by which this occurs is unknown. In this study, two groups consisting of 3140 fish that were raised in either aerated (normoxia) or non-aerated pond (nocturnal hypoxia). During grow out, fish were sampled five times to determine individual body weight (BW) gains. We applied a genome-wide association study to identify SNPs and genes associated with the hypoxic and normoxic environments in the 16th generation of a Genetically Improved Farmed Tilapia population.ResultsIn the hypoxic environment, 36 SNPs associated with at least one of the five body weight measurements (BW1 till BW5), of which six, located between 19.48 Mb and 21.04 Mb on Linkage group (LG) 8, were significant for body weight in the early growth stage (BW1 to BW2). Further significant associations were found for BW in the later growth stage (BW3 to BW5), located on LG1 and LG8. Analysis of genes within the candidate genomic region suggested that MAPK and VEGF signalling were significantly involved in the later growth stage under the hypoxic environment. Well-known hypoxia-regulated genes such as igf1rb, rora, efna3 and aurk were also associated with growth in the later stage in the hypoxic environment. Conversely, 13 linkage groups containing 29 unique significant and suggestive SNPs were found across the whole growth period under the normoxic environment. A meta-analysis showed that 33 SNPs were significantly associated with BW across the two environments, indicating a shared effect independent of hypoxic or normoxic environment. Functional pathways were involved in nervous system development and organ growth in the early stage, and oocyte maturation in the later stage.ConclusionsThere are clear genotype-growth associations in both normoxic and hypoxic environments, although genome architecture involved changed over the growing period, indicating a transition in metabolism along the way. The involvement of pathways important in hypoxia especially at the later growth stage indicates a genotype-by-environment interaction, in which MAPK and VEGF signalling are important components.

Intrauterine Growth Restriction Promotes Postnatal Airway Hyperresponsiveness Independent of Allergic Disease

Introduction: Intrauterine growth restriction (IUGR) is associated with asthma. Murine models of IUGR have altered airway responsiveness in the absence of any inflammatory exposure. Given that a primary feature of asthma is airway inflammation, IUGR-affected individuals may develop more substantial respiratory impairment if subsequently exposed to an allergen. This study used a maternal hypoxia-induced mouse model of IUGR to determine the combined effects of IUGR and allergy on airway responsiveness.Methods: Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O2) from gestational day (GD) 11-GD 17.5 (IUGR group; term = GD 21). Following hypoxic exposure, mice were returned to a normoxic environment (21% O2). A second group of pregnant mice were housed under normoxic conditions throughout pregnancy (Control). All offspring were sensitized to ovalbumin (OVA) and assigned to one of four treatment groups: Control – normoxic and saline challenge; IUGR – hypoxic and saline challenge; Allergy – normoxic and OVA challenge; and IUGR + Allergy – hypoxic and OVA challenge. At 8 weeks of age, and 24 h post-aerosol challenge, mice were tracheostomised for methacholine challenge and assessment of lung mechanics by the forced oscillation technique, and lungs subsequently fixed for morphometry.Results: IUGR offspring were lighter than Control at birth and in adulthood. Both Allergy and IUGR independently increased airway resistance after methacholine challenge. The IUGR group also exhibited an exaggerated increase in tissue damping and elastance after methacholine challenge compared with Control. However, there was no incremental effect on airway responsiveness in the combined IUGR + Allergy group. There was no impact of IUGR or Allergy on airway structure and no effect of sex on any outcome.Conclusion: IUGR and aeroallergen independently increased bronchoconstrictor response, but when combined the pathophysiology was not worsened. Findings suggest that an association between IUGR and asthma is mediated by baseline airway responsiveness rather than susceptibility to allergen.

Effects of Intermittent Normoxia on Chronic Hypoxic Pulmonary Hypertension and Right Ventricular Hypertrophy in Rats.

Liu, Chunlei, Xu Chen, Ge Guo, Xiang Xu, Xin Li, Qingxia Wei, Yanying Shen, Hanlu Li, Jianxiu Hao, Ya Ping Tian, and Kunlun He. Effects of intermittent normoxia on chronic hypoxic pulmonary hypertension and right ventricular hypertrophy in rats. High Alt Med Biol. 22: 184-192, 2021. Background: Individuals with chronically low arterial oxygen tension owing to high altitude develop elevated rates of pulmonary hypertension (PH) and right ventricular (RV) hypertrophy. However, the effects of the frequency and duration of normoxic exposure on PH and RV hypertrophy have not been adequately assessed; thus, we aimed to analyze the same. Materials and Methods: PH and RV hypertrophy were induced in 60 rats using a hypobaric chamber. Of these 60 rats, every 10 were exposed to normoxic conditions for 30 minutes once (1T/D), three times (3T/D), or five times daily (5T/D), or for one 150-minute recovery daily (1LT/D). Furthermore, 10 rats were housed in a normoxic environment, and another 10 were subjected to continuous hypoxia. After 4 weeks, hemodynamic measurements were recorded, and the hearts were harvested for pathomorphological observations. Results: Average pulmonary arterial pressures (PAP) of control rats and those exposed to hypobaric hypoxia were 14.1 and 32.3 mmHg, respectively. After 30 minutes of exposure to normoxia 3T/D, 5T/D, or 1LT/D, PAP values were reduced to 27.1, 27.9, or 26.8 mmHg, respectively. Four weeks of hypoxic exposure elevated the RV/heart weight (HW) ratios, while exposure to normoxia 3T/D, 5T/D, and 1LT/D significantly reduced RV/HW. In addition, exposure to normoxia 3T/D, 5T/D, 1LT/D reduced the percentage wall thickness of the pulmonary artery as well as the hypertrophy indices of atrial natriuretic peptide, brain natriuretic peptide, and myosin heavy chain 7 (MYH-7). Conclusions: Thirty-minute exposure to normoxic conditions of 3T/D, 5T/D, or 1LT/D effectively ameliorates PH and RV thickening.

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds.

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young’s modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Use of photosynthetic transgenic cyanobacteria to promote lymphangiogenesis in scaffolds for dermal regeneration

Impaired wound healing represents an unsolved medical need with a high impact on patients´ quality of life and global health care. Even though its causes are diverse, ischemic-hypoxic conditions and exacerbated inflammation are shared pathological features responsible for obstructing tissue restoration. In line with this, it has been suggested that promoting a normoxic pro-regenerative environment and accelerating inflammation resolution, by reinstating the lymphatic fluid transport, could allow the wound healing process to be resumed. Our group was first to demonstrate the functional use of scaffolds seeded with photosynthetic microorganisms to supply tissues with oxygen. Moreover, we previously proposed a photosynthetic gene therapy strategy to create scaffolds that deliver other therapeutic molecules, such as recombinant human growth factors into the wound area. In the present work, we introduce the use of transgenic Synechococcus sp. PCC 7002 cyanobacteria (SynHA), which can produce oxygen and lymphangiogenic hyaluronic acid, in photosynthetic biomaterials. We show that the co-culture of lymphatic endothelial cells with SynHA promotes their survival and proliferation under hypoxic conditions. Also, hyaluronic acid secreted by the cyanobacteria enhanced their lymphangiogenic potential as shown by changes to their gene expression profile, the presence of lymphangiogenic protein markers and their capacity to build lymph vessel tubes. Finally, by seeding SynHA into collagen-based dermal regeneration materials, we developed a viable photosynthetic scaffold that promotes lymphangiogenesis in vitro under hypoxic conditions. The results obtained in this study lay the groundwork for future tissue engineering applications using transgenic cyanobacteria that could become a therapeutic alternative for chronic wound treatment. Statement of significanceIn this study, we introduce the use of transgenic Synechococcus sp. PCC 7002 (SynHA) cyanobacteria, which were genetically engineered to produce hyaluronic acid, to create lymphangiogenic photosynthetic scaffolds for dermal regeneration. Our results confirmed that SynHA cyanobacteria maintain their photosynthetic capacity under standard human cell culture conditions and efficiently proliferate when seeded inside fibrin-collagen scaffolds. Moreover, we show that SynHA supported the viability of co-cultured lymphatic endothelial cells (LECs) under hypoxic conditions by providing them with photosynthetic-derived oxygen, while cyanobacteria-derived hyaluronic acid stimulated the lymphangiogenic capacity of LECs. Since tissue hypoxia and impaired lymphatic drainage are two key factors that directly affect wound healing, our results suggest that lymphangiogenic photosynthetic biomaterials could become a treatment option for chronic wound management.

Metal-organic frameworks-derived bimetallic nanozyme platform enhances cytotoxic effect of photodynamic therapy in hypoxic cancer cells

Photodynamic therapy (PDT) is increasingly accepted as a cancer treatment because it can target the tumor precisely and treat it noninvasively. The therapeutic effect of PDT is generally affected by three parameters: the light, the photosensitizers (PSs), and the local oxygen, where the performance of the PSs with light is greatly affected by the availability of local oxygen. However, hypoxia is one of the typical characteristics of the microenvironment in solid tumors, which renders the efficacy of PDT in cancer treatment. Here, we introduced a novel nanozyme platform, which composed of metal-organic frameworks (MOF) derived materials and could directly load the PSs. The nanozyme could generate oxygen by catalyzing H2O2, which enhanced the production of reactive oxygen species (ROS) and resulted in an improved cytotoxic effect of the PSs. We showed that, especially under the hypoxic environment, this nanozyme could alleviate the hypoxic situation by generating oxygen in the H2O2 solution and further improved the therapeutic effect of PDT consequently. In conclusion, this nanozyme platform allows the loading of PSs with ease and can catalyze H2O2 to generate oxygen to enhance the effect of PDT for cancer treatment in both normoxic and hypoxic environments.

Variations of Time Irreversibility of Heart Rate Variability Under Normobaric Hypoxic Exposure.

In the field of biomedicine, time irreversibility is used to describe how imbalanced and asymmetric biological signals are. As an important feature of signals, the direction of time is always ignored. To find out the variation regularity of time irreversibility of heart rate variability (HRV) in the initial stage of hypoxic exposure, the present study implemented 2 h acute normobaric hypoxic exposure on six young subjects who have no plateau or hypoxia experiences; oxygen concentration was set as 12.9%. Electrocardiogram (ECG) signals were recorded in the whole process and RR interval sequences were extracted. Mathematical operations were executed to transform the difference of adjacent RR intervals into proportion and distance with delay time to conduct time irreversibility analysis of HRV. The same calculating method was implemented on six items randomly picked out from the MIT-BIH normal sinus rhythm database as a control group. Results show that variation of time irreversibility of HRV in a hypoxic environment is different from that in a normoxic environment, time irreversibility indices of a hypoxic group decreases continually at a delay time of 1 and 2, and indices curves of time irreversibility gradually tend to be steady and gather with each other at a delay time of 3 or 4. The control group shows no consistent tendency no matter what the delay time is in the range of 1–4. Our study indicates that in short-time hypoxic exposure, as hypoxic time goes by, regulation of the cardiovascular autonomic nervous system weakens; regulation times and intensity of sympathetic and parasympathetic nerves tend to be equal.

Effectiveness of Physical Fitness Training of Athletes Based On Parameter Bayesian Estimation

In this paper, 64 national first-class athletes in speed skating and short-track speed skating were selected to participate in hypoxia training. In the study, athletes participated in a total of eight weeks of training, measured and analyzed the physiological indexes of athletes in normoxic environment and hypoxic environment. In the study, data analysis combined with machine learning was used to obtain the conversion formula of athletes’ physiological indexes in hypoxia training and normoxia training. The experimental results show that the simulated hypoxia environment of 2300m is effective for athletes participating in both sports, in which the effect of short track speed skating is obvious, and the effect of speed skating is weak. Through the analysis, it is found that female athletes are more easily affected by hypoxia training. Relatively speaking, women who take part in the same hypoxia training get more benefits. The physiological indexes in normoxic environment and hypoxic environment are used in machine learning, and the transformation formulas of various physiological indexes under the environmental conditions in this study are successfully obtained. It is proved that the error between the hypoxia physiological index estimated by the transformation formula and the actual physiological index is small, which shows that the transformation formula is more accurate. This proves that hypoxia training can be estimated by means of machine learning to obtain more accurate data guidance, which has a strong practical significance for coaches to guide athletes’ training.

Blood oxidative stress and post-exercise recovery are unaffected byhypobaric and hypoxic environments

ABSTRACT Hypobaria and hypoxia exert independent effects on oxidative stress during exercise, while combined effectson the post-exercise recovery period remain unclear.Accordingly, this study examined the recovery period during lab-simulated hypoxic and hypobaric conditions following exercise-induced oxidative stress. Participants (n=13) performed 60-minutes of cycling (70% watts max) in a normobaric normoxic environment followed by a four-hour recovery under three conditions; 1000m normobaric normoxia (NN, 675mmHg), 4400m normobaric hypoxia (NH, 675mmHg), or 4400m hypobaric hypoxia (HH, 440mmHg). Blood samples collected at Pre, Post, 2-Hours (2-HR), and 4-Hours (4-HR) post-exercise were analyzed fora potential increase in biochemical modifications of proteins(protein carbonyls, PC; 3-nitrotyrosines, 3NT) lipids (lipid hydroperoxides, LOOH; 8-isoprostanes, 8-ISO), and antioxidant capacity (FRAP, TEAC). Gene transcripts (EPAS, HMOX1, SOD2, NFE2L2) were quantified by qRT-PCR from muscle biopsies taken Pre and Post exercise. Hypoxia and hypobaria had no effect throughout recovery. Post-exercise TEAC (p=0.041), FRAP (p=0.013), and 8-ISO (p=0.044) increased, while PC (p=0.002) and 3-NT (p=0.032) were decreased. LOOH was lower in Post (p=0.018) NH trial samples. Exercise-dependent increases occurred in NFE2L2 (p=0.003), HMXO1 (p<0.001), SOD2 (p=0.046), and EPAS (p=0.038). Exercise recovery under conditions of NH and HH did not impact blood oxidative stress or redox-sensitive gene transcripts.

Cascade Reactions by Nitric Oxide and Hydrogen Radical for Anti-Hypoxia Photodynamic Therapy Using an Activatable Photosensitizer.

Organelle-targeted activatable photosensitizers are attractive to improve the specificity and controllability of photodynamic therapy (PDT), however, they suffer from a big problem in the photoactivity under both normoxia and hypoxia due to the limited diversity of phototoxic species (mainly reactive oxygen species). Herein, by effectively photocaging a π-conjugated donor-acceptor (D-A) structure with an N-nitrosamine substituent, we established a unimolecular glutathione and light coactivatable photosensitizer, which achieved its high performance PDT effect by targeting mitochondria through both type I and type II (dual type) reactions as well as secondary radicals-participating reactions. Of peculiar interest, hydrogen radical (H•) was detected by electron spin resonance technique. The generation pathway of H• via reduction of proton and its role in type I reaction were discussed. We demonstrated that the synergistic effect of multiple reactive species originated from tandem cascade reactions comprising reduction of O2 by H• to form O2•-/HO2• and downstream reaction of O2•- with •NO to yield ONOO-. With a relatively large two-photon absorption cross section for photoexcitation in the near-infrared region (166 ± 22 GM at 800 nm) and fluorogenic property, the new photosensitizing system is very promising for broad biomedical applications, particularly low-light dose PDT, in both normoxic and hypoxic environments.

Extracellular Vesicles Are Key Regulators of Tumor Neovasculature.

Tumor progression involves a series of biologically important steps in which the crosstalk between cancer cells and the surrounding environment is an important issue. Angiogenesis is a key tumorigenic phenomenon for cancer progression. Tumor-related extracellular vesicles (EVs) modulate the tumor microenvironment (TME) through cell-to-cell communication. Tumor cells in a hypoxic TME release more EVs than cells in a normoxic environment due to uncontrollable tumor proliferation. Tumor-derived EVs in the TME influence endothelial cells (ECs), which then play multiple roles, contributing to tumor angiogenesis, loss of the endothelial vascular barrier by binding to ECs, and subsequent endothelial-to-mesenchymal transition. In contrast, they also indirectly induce tumor angiogenesis through the phenotype switching of various cells into cancer-associated fibroblasts, the activation of tumor-associated ECs and platelets, and remodeling of the extracellular matrix. Here, we review current knowledge regarding the involvement of EVs in tumor vascular-related cancer progression.

A Deep Learning Framework for Sickle Cell Disease Microfluidic Biomarker Assays

Introduction: Vaso-occlusive crises (VOCs) are a leading cause of morbidity and early mortality in individuals with sickle cell disease (SCD). These crises are triggered by sickle red blood cell (sRBC) aggregation in blood vessels and are influenced by factors such as enhanced sRBC and white blood cell (WBC) adhesion to inflamed endothelium. Advances in microfluidic biomarker assays (i.e., SCD Biochip systems) have led to clinical studies of blood cell adhesion onto endothelial proteins, including, fibronectin, laminin, P-selectin, ICAM-1, functionalized in microchannels. These microfluidic assays allow mimicking the physiological aspects of human microvasculature and help characterize biomechanical properties of adhered sRBCs under flow. However, analysis of the microfluidic biomarker assay data has so far relied on manual cell counting and exhaustive visual morphological characterization of cells by trained personnel. Integrating deep learning algorithms with microscopic imaging of adhesion protein functionalized microfluidic channels can accelerate and standardize accurate classification of blood cells in microfluidic biomarker assays. Here we present a deep learning approach into a general-purpose analytical tool covering a wide range of conditions: channels functionalized with different proteins (laminin or P-selectin), with varying degrees of adhesion by both sRBCs and WBCs, and in both normoxic and hypoxic environments. Methods: Our neural networks were trained on a repository of manually labeled SCD Biochip microfluidic biomarker assay whole channel images. Each channel contained adhered cells pertaining to clinical whole blood under constant shear stress of 0.1 Pa, mimicking physiological levels in post-capillary venules. The machine learning (ML) framework consists of two phases: Phase I segments pixels belonging to blood cells adhered to the microfluidic channel surface, while Phase II associates pixel clusters with specific cell types (sRBCs or WBCs). Phase I is implemented through an ensemble of seven generative fully convolutional neural networks, and Phase II is an ensemble of five neural networks based on a Resnet50 backbone. Each pixel cluster is given a probability of belonging to one of three classes: adhered sRBC, adhered WBC, or non-adhered / other. Results and Discussion: We applied our trained ML framework to 107 novel whole channel images not used during training and compared the results against counts from human experts. As seen in Fig. 1A, there was excellent agreement in counts across all protein and cell types investigated: sRBCs adhered to laminin, sRBCs adhered to P-selectin, and WBCs adhered to P-selectin. Not only was the approach able to handle surfaces functionalized with different proteins, but it also performed well for high cell density images (up to 5000 cells per image) in both normoxic and hypoxic conditions (Fig. 1B). The average uncertainty for the ML counts, obtained from accuracy metrics on the test dataset, was 3%. This uncertainty is a significant improvement on the 20% average uncertainty of the human counts, estimated from the variance in repeated manual analyses of the images. Moreover, manual classification of each image may take up to 2 hours, versus about 6 minutes per image for the ML analysis. Thus, ML provides greater consistency in the classification at a fraction of the processing time. To assess which features the network used to distinguish adhered cells, we generated class activation maps (Fig. 1C-E). These heat maps indicate the regions of focus for the algorithm in making each classification decision. Intriguingly, the highlighted features were similar to those used by human experts: the dimple in partially sickled RBCs, the sharp endpoints for highly sickled RBCs, and the uniform curvature of the WBCs. Overall the robust performance of the ML approach in our study sets the stage for generalizing it to other endothelial proteins and experimental conditions, a first step toward a universal microfluidic ML framework targeting blood disorders. Such a framework would not only be able to integrate advanced biophysical characterization into fast, point-of-care diagnostic devices, but also provide a standardized and reliable way of monitoring patients undergoing targeted therapies and curative interventions, including, stem cell and gene-based therapies for SCD. Disclosures Gurkan: Dx Now Inc.: Patents &amp; Royalties; Xatek Inc.: Patents &amp; Royalties; BioChip Labs: Patents &amp; Royalties; Hemex Health, Inc.: Consultancy, Current Employment, Patents &amp; Royalties, Research Funding.

Sex differences in modulation of fetoplacental vascular resistance in growth-restricted mouse fetuses following betamethasone administration: comparisons with human fetuses

Maternally administered corticosteroids are routinely used to accelerate fetal lung maturation in pregnancies at risk of early preterm delivery. Although, among the subgroup with growth restriction, a majority show a temporary improvement in umbilical artery Doppler waveforms that may be sustained up to 7 days, a minority will acutely decompensate in response to corticosteroids in association with deteriorating umbilical and fetal Doppler waveforms. The basis for such acute Doppler changes is presently unknown. Our group has developed a noninvasive ultrasound methodology to measure wave reflections in the umbilical artery and have established that wave reflection metrics are sensitive to structural changes in the placental vasculature and to acute changes in vascular tone. Using this approach, we demonstrated in healthy pregnant mice that fetoplacental vascular resistance decreased in betamethasone-treated mice compared with saline-treated controls. This study aimed to investigate the effects of betamethasone administration on the wave reflection metrics in a mouse model of fetal growth restriction and to compare these findings with equivalent measurements in human fetuses. Pregnant CD-1 mice were housed from embryonic day 14.5 to embryonic day 17.5 in either a normoxic (21% O2, n=24) or hypoxic environment (11% O2, n=22), the latter being an established mouse model of fetal growth restriction. To investigate the effect of maternally administered betamethasone on the fetoplacental vasculature, ultrasound imaging was performed at baseline and 4 hours after treatment (either betamethasone or sterile saline). Umbilical artery wave reflection metrics were compared between the groups and for the effect of fetal sex. In addition, a cohort of 10 pregnant women with elevated umbilical artery pulsatility index and evidence of fetal growth restriction and 6 controls were imaged before and after corticosteroid administration. In the mouse model, after betamethasone administration, the female fetuses from the hypoxia group showed a 15% increase in umbilical artery diameter, a 98% increase in umbilical artery blood flow, and a 27% decrease in umbilical artery reflection coefficient, whereas the males from the hypoxia group showed no substantial changes. In agreement with our mouse findings, umbilical artery reflections were found to be larger in human growth-restricted fetuses than controls in women at risk of preterm birth. Our studies provide insight into the mechanism whereby the human growth-restricted fetus may exhibit a temporary favorable fetoplacental vascular response to maternally administered corticosteroids. Further investigations are needed to understand why the male growth-restricted fetus seems unable to mount this favorable vascular response.

Intrauterine growth restriction predisposes to airway inflammation without disruption of epithelial integrity in postnatal male mice.

Evidence from animal models demonstrate that intrauterine growth restriction (IUGR) alters airway structure and function which may affect susceptibility to disease. Airway inflammation and dysregulated epithelial barrier properties are features of asthma which have not been examined in the context of IUGR. This study used a maternal hypoxia-induced IUGR mouse model to assess lung-specific and systemic inflammation and airway epithelial tight junctions (TJs) protein expression. Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O2) from gestational day (GD) 11 to 17.5 (IUGR group; term, GD 21). Following hypoxic exposure, mice were returned to a normoxic environment (21% O2). A Control group was housed under normoxic conditions throughout pregnancy. Offspring weights were recorded at 2 and 8 weeks of age and euthanized for bronchoalveolar lavage (BAL) and peritoneal cavity fluid collection for inflammatory cells counts. From a separate group of mice, right lungs were collected for Western blotting of TJs proteins. IUGR offspring had greater inflammatory cells in the BAL fluid but not in peritoneal fluid compared with Controls. At 8 weeks of age, interleukin (IL)-2, IL-13, and eotaxin concentrations were higher in male IUGR compared with male Control offspring but not in females. IUGR had no effect on TJs protein expression. Maternal hypoxia-induced IUGR increases inflammatory cells in the BAL fluid of IUGR offspring with no difference in TJs protein expression. Increased cytokine release, specific to the lungs of IUGR male offspring, indicates that both IUGR and sex can influence susceptibility to airway disease.

Insulin ameliorates hypoxia-induced autophagy, endoplasmic reticular stress and apoptosis of myocardial cells: In vitro and ex vivo models

Whether and how insulin counteracts the cytotoxic effects of hypoxia and improves cardiomyocyte viability remains unclear. To achieve this aim, cultured neonatal rat cardiomyocytes pretreated with vehicle or 1 μM insulin were exposed to either normoxic or hypoxia environment for up to 24 h. Cell viability was monitored and cellular apoptosis as well as necrosis, indexes of autophagy, endoplasmic reticular (ER) stress, and expressions of specific relevant mediators of the signaling pathways of autophagy were also assessed. Hypoxia impaired cell viability, induced autophagy, triggered apoptosis, activated ER stress pathway-associated apoptotic responses along with downstream pro-apoptotic transcriptional factor C/EBP homologous protein (CHOP), and increased apoptosis of myocardial cells. On the other hand, insulin pretreatment effectively ameliorated autophagy via PI3–K/Akt signaling pathway, suppressed ER stress, and prevented hypoxia-induced cellular apoptosis. In an ex vivo study, isolated rat hearts were pre-treated in some cases with insulin and subjected to proximal left coronary artery ligation to induce acute myocardial ischemia. Coronary ligation-induced acute ischemia upregulated glucose-related protein 78 (GRP78) and triggered cellular apoptosis in the jeopardized myocardium. Conversely, insulin pretreatment suppressed these hypoxia-related cytotoxic events and reduced myocardial infarct size by up to 15.2%. In conclusion, hypoxia impedes cell viability through triggering autophagy, ER stress and apoptosis, whereas insulin pretreatment effectively prevents these cytotoxic actions of hypoxia, preserves myocardial cell viability and reduces myocardial infarct size. These results indicated the cytoprotective mechanism of insulin against the insult of hypoxia may justify insulin as a therapeutic option for patients with acute myocardial infarction.