We examined the impact of moderate hypoxia (HYPO) on muscle activation during incremental exercise matched for both absolute and equivalent relative intensity. Fifteen active subjects (10 males, 5 females) completed two ramp incremental test and two step tests in normoxia (NORM; =0.209) and HYPO ( ≈ 0.135) in counterbalanced order. The respiratory compensation point (RCP) determined from ramp testing was used to normalize relative intensity during step testing, which included a final stage to task failure (TF) above RCP. Electromyography (EMG) was recorded for rectus femoris (RF), vastus lateralis (VL) and vastus medialis (VM), and normalized to a pre-test maximal sprint effort. Linear mixed modelling was used to examine fixed effects of condition (NORM, HYPO) and intensity (absolute, relative) on EMG activity. During the ramp test, HYPO significantly reduced (∼13%), PPO (∼15%), and power at RCP (∼16%). EMG breakpoints occurred at lower absolute intensity in HYPO for RF and VL. When matched for relative intensity, muscle activity was lower in HYPO for VM and VL, but not RF. EMG activity at TF revealed a similar pattern whereby a strong association to absolute power was present regardless of test protocol or . These results suggest that altered relative metabolic stress has a negligible impact on muscle activation at work rates below the RCP. For exercise in the severe domain our data aligns with the theory that muscle activation is not critically regulated to a given level at TF, but appears to be task-specific and independent of oxygen availability.

Maternal obesity increases the risk of preterm delivery and rapid transition of offspring from a hypoxemic environment to a normal or elevated oxygen environment, especially if the baby receives oxygen therapy. Maternal obesity may also increase offspring risk of developing hypertension. Thus, we examined whether neonatal hyperoxia (HO) leads to elevated blood pressure (BP) in offspring from lean mothers and exacerbates adverse impact of maternal obesity on offspring BP regulation. Male and female Sprague-Dawley offspring from lean and high-fat diet-fed obese mothers (n = 12-18 mothers/group) were exposed to room air (∼21% O2) or HO (80% O2) between postnatal days P3 and P10 and then returned to room air. At 12 wk of age, offspring were instrumented with telemetry probes to measure BP and heart rate (HR). Contrary to our hypothesis, neonatal HO was associated with lower BP compared with control offspring from lean mothers (males: 105 ± 1 vs. 111 ± 1 mmHg; females: 102 ± 0.4 vs. 108 ± 0.4 mmHg) and also reduced BP and HR in hypertensive obese offspring from obese mothers (males: 117 ± 1 vs. 123 ± 1 mmHg and 351 ± 4 vs. 358 ± 5 beats/min; females: 113 ± 1 vs. 116 ± 1 mmHg and 376 ± 2 vs. 390 ± 4 beats/min). In lean offspring from lean mothers, neonatal HO was associated with reduced +dP/dtmax, whereas in obese offspring from obese mothers, HO attenuated cardiac dysfunction when compared with obese offspring not submitted to HO. These results suggest that exposure to HO in early postnatal life is not associated with elevated BP in early adulthood and it does not exacerbate the hypertensive effects of maternal obesity on offspring BP regulation.NEW & NOTEWORTHY Maternal obesity increases risk for preterm birth and neonatal oxygen exposure. We tested whether hyperoxia (80% O2, P3-P10) worsens maternal obesity-induced hypertension. At 14 wk, BP measured by telemetry showed that hyperoxia unexpectedly lowered BP in lean and obese offspring and attenuated cardiac dysfunction in obese offspring. These findings indicate that neonatal hyperoxia does not exacerbate maternal obesity-induced hypertension and may mitigate early cardiac dysfunction.

BackgroundBronchopulmonary dysplasia (BPD) is a common respiratory complication in premature infants, in which pulmonary fibrosis will continue to lead to the long-term impairment of pulmonary function in children with BPD and seriously affect the quality of life. This study hopes to explore the changing trend of pulmonary fibrosis characteristics of chronic hyperoxia lung injury.MethodsNewborn rats were randomly assigned to a room air (RA) group or a hyperoxia (HO) group and sampled at postnatal days (P) 3, 7, 14, 21, 28, and 42. Lung morphology was assessed using histological staining to evaluate alveolar development, collagen volume fraction (CVF), and myofibroblast distribution. Western blotting was used to measure protein expression levels of surfactant protein C (SPC), podoplanin (PDPN), cluster of differentiation 31 (CD31), and α-smooth muscle actin (α-SMA). Expression levels of fibrosis-related markers [α-SMA, Collagen I (Col I)] were evaluated at both protein and messenger ribonucleic acid (mRNA) levels using Western blot and quantitative real-time polymerase chain reaction (RT-qPCR), respectively. Additional RT-qPCR analysis was performed for fibronectin and connective tissue growth factor (CTGF).ResultsAt P14, HO rats exhibited decreased radical alveolar count (RAC), increased mean linear intercept (MLI), and elevated CVF compared to RA rats. SPC, CD31, and PDPN protein levels were reduced, while α-SMA increased. Collagen deposition progressively increased, peaking at P42. Col I expression rose significantly at P21 and P42. Fibronectin mRNA peaked at P21, while CTGF mRNA increased from P7 and remained elevated through P21.ConclusionsPulmonary fibrosis in chronic hyperoxia-induced lung injury emerged by P14, peaked around P21, and stabilized thereafter. These findings offer insight into the temporal dynamics of fibrosis development in BPD and may help guide timing for therapeutic intervention.

Objective: Hypoxia (HPX) increases the amount of Ca2+ influx, apoptosis, and harmful free reactive oxygen species (ROS) in the brain and neurons. Resveratrol (RES) has been shown to reduce these increases in ROS-damaged neuronal cells by inhibiting voltage-gated Ca2+ channels. The aim of the study was to ascertain whether RES could also inhibit the elevated ROS and apoptosis induced by HPX in SH-SY5Y glioblastoma cells via inhibiting TRPA1. Materials and Methods: In the SH-SY5Y, four primary groups were induced as control, RES (50 µM for 24h), HPX (200 µM CoCl2 for 24h), and HPX + RES. Results: While the incubations of the TRPA1 antagonist (AP-18) and RES decreased the HPX-mediated upregulations of apoptotic (caspase -3, -8, and -9) and oxidants (ROS, mitochondrial dysfunction, and lipid peroxidation) concentrations, the TRPA1 agonist (cinnamaldehyde) stimulation further increased these concentrations. The RES increased viable cell percentage, glutathione concentration, and glutathione peroxidase activity, all of which were diminished by HPX. Conclusions: The concentrations of HPX-induced neuronal apoptosis and mitochondrial oxidative stress were reduced by RES treatment through TRPA1 inhibition. It seems that RES is a potential treatment option for HPX-induced mitochondrial oxidative neuronal injury.

Hyperoxia (HO) and mechanical ventilation (MV) are the mainstay of treatment for patients with acute respiratory failure, but both interventions can also accelerate further lung injury, highlighting the need for better therapeutic approaches. We previously found that HO decreases epithelial TREK-1 expression and promotes epithelial inflammation, but the consequences of TREK-1 deficiency in a clinically relevant system of combined HO + ST (stretch) exposure remain unknown. We found that in both mouse lung tissue and primary human alveolar epithelial cells, HO + ST downregulates TREK-1 protein levels. The injurious consequences of TREK-1 downregulation are evidenced in alveolar epithelial cells following pharmacological and genetic TREK-1 inhibition and in lungs of TREK-1 KO mice by potentiation of HO + ST-induced cytosolic ROS production, caspase-8 and caspase-1 activation, IL-1β production, and MIP-1α, and CXCL-10/IP-10 secretion. In addition, HO + MV-exposed TREK-1 KO mice show increased histological lung injury scores, total cell, macrophage, and neutrophil counts in the bronchoalveolar lavage fluid (BALF). Mechanistically, HO + ST depolarized the epithelial electrical membrane potential (Em) and raised iCa2+ levels, which was potentiated after pharmacological and genetic TREK-1 inhibition. Both Ca2+ influx through voltage-gated Ca2+ channels and Ca2+ release from intracellular stores increased iCa2+ levels following TREK-1 inhibition. Intratracheal administration of two structurally different pharmacological TREK-1 activators (ML335, BL1249) improved HO + ST-induced BALF total and differential cell counts, total protein levels, ROS production, caspase-8 and capase-1 production, and cytokine concentrations. Therefore, these findings highlight TREK-1 as new potential target for intervention against HO + ST/MV-induced lung and epithelial injury and lay the groundwork for future rational drug development.NEW & NOTEWORTHY No targeted interventions exist that improve the outcomes of patients with acute lung injury/ARDS. A few studies investigated Na+ and Ca2+ channels/transporters for potential therapeutic intervention but with limited translational success. This study highlights the regulatory role of TREK-1 K+ channels during HO+stretch/mechanical ventilation-induced lung injury in ROS production, caspase activation, cytokine secretion, and explores the underlying TREK-1-mediated signaling mechanisms. These preclinical findings lay the groundwork for future rational drug design targeting TREK-1 channels.

Bronchopulmonary dysplasia (BPD) associated pulmonary hypertension (PH) or BPD-PH is a lung disease of infants with significant morbidity. Adrenomedullin (Adm) is an angiogenic peptide that signals through calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2 (RAMP2). Adm deficiency potentiates hyperoxia-induced experimental BPD-PH in mice; however, whether Adm overexpression can mitigate this lung disease is unclear. Thus, we tested the hypothesis that Adm overexpression attenuates hyperoxia (HO)-induced murine experimental BPD-PH by using a novel transgenic mouse that overexpresses Adm globally (Admhi/hi mice). One-day-old Admhi/hi mice or their wild-type littermates (Adm+/+ mice) were exposed to HO ([Formula: see text] 70%) for 14 days and allowed to recover in normoxia (NO, [Formula: see text] 21%) for an additional 14 days. Controls were maintained in NO for 28 days. On postnatal day (P) 14, we harvested the lungs to determine the extent of Adm expression and apoptosis. On P28, we quantified alveolarization, lung vascularization, and PH. HO-exposed Adm+/+ mice demonstrated increased lung apoptosis, decreased alveolarization and lung vascularization, and indices of PH, indicating that neonatal HO exposure causes BPD-PH. However, Adm overexpression attenuated experimental BPD-PH, as evident by the decreased extent of hyperoxia-induced lung apoptosis and inflammation, alveolar and vascular simplification, pulmonary vascular remodeling, and PH in Admhi/hi mice than in Adm+/+ mice. Collectively, our results demonstrate that Adm overexpression attenuates HO-induced murine experimental BPD-PH, emphasizing the therapeutic potential of Adm for BPD-PH in preterm infants.NEW & NOTEWORTHY The deficiency of the proangiogenic peptide, adrenomedullin (Adm), exacerbates the severe infantile lung disorder, bronchopulmonary dysplasia-associated pulmonary hypertension (BPD-PH), in mice. However, whether Adm therapy can mitigate this disease is unclear. Our study, conducted with a rigorous methodology, suggests a potential solution. Using a novel mouse that overexpresses Adm to overcome the pharmacological limitations of the peptide, we demonstrate that Adm can mitigate this disorder, highlighting the therapeutic potential of Adm for human BPD-PH.

Preclinical studies provided convincing evidence that umbilical cord derived mesenchymal stem cells (UC-MSC) prevent lung injury and promote lung regeneration. We hypothesized that cyclic mechanical stretch (CMS) and hyperoxia (HOX) during mechanical ventilation account for their limited therapeutic efficacy within the clinics. UC-MSC cultures were subjected to CMS and HOX and evaluated for proliferation, cell viability and further functional properties. Reversibility of the phenotype changes was evaluated after recovery in room air following these exposures. CMS and HOX compromised cell viability and proliferation, altered phenotypic characteristics, particularly PDGFRα expression, and induced cellular senescence in UC-MSC. Effects were most pronounced for CMS plus HOX. The alterations of UC-MSC were mediated by p21 accumulation. As inhibition of p21 aggravated cell death of UC-MSC, the results indicated a cell defense mechanism to ensure survival. This assumption was underpinned by the principal reversibility of the phenotype alterations and regrowth after removal of CMS and HOX. But prolonged strongest exposures resulted in definite phenotype changes. CMS and HOX have comparable effects on UC-MSC as described for lung resident MSC. Our results explain their timely limited presence in the diseased lung after therapeutic application. Future research should therefore focus on their repetitive application.

Early Alzheimer's disease (AD) is characterized by anti-inflammatory microglial responses to the beta amyloid peptide (Aβ), which later switch to pro-inflammatory. Such transition is relevant to disease progression and can be affected by concurrent insults, such as hypoxia (HY). This study explored whether a mild hypoxic stimulus could anticipate the microglial phenotypic switch, focusing in particular on involvement of SIRT1 and mitochondrial function. HMC3 human microglia were polarized to an anti-inflammatory phenotype by 3h of exposure to 0.2μM of Aβ42 to mimic early AD and transferred to a hypoxic chamber with 3% of O2 for 1h. Effects on microglial activation were investigated by analysis of the SIRT1-BDNF axis activation and enzymatic and ELISA assays of inflammatory markers. Mitochondrial function and morphology were analyzed by high resolution respirometry and laser scanning confocal microscopy. Hypoxia (HY) prevented the Aβ42-induced early induction of SIRT1 translocation and BDNF release and significantly increased caspase 1 and NF-kB activity. Moreover, mitochondrial oxygen flows evaluated by high resolution respirometry were significantly reduced, while mitochondrial area, perimeter and branching were increased by Aβ42 + HY, compared to Aβ alone. These changes were contrasted by both melatonin (1μM) and naringenin (10μM), natural substances able to induce SIRT1. However, use of the selective SIRT1 inhibitor EX-527 (5μM) suggested only a partial involvement for SIRT1 in the observed effects, prevalent for naringenin. Our results suggest that mild hypoxic insults during early asymptomatic stages of AD can pose as a risk factor for an accelerated progression of the disease and show the benefits of SIRT1 induction strategies, including use of natural substances like melatonin and naringenin.

Untargeted LC-MS/MS- based metabolomics profiling of colorectal cancer cell lines reveals potential hypoxia-associated biomarkers.

The chronic hypoxia of high-altitude (HA, >2500 m) residence reduces uterine artery (UtA) blood flow, contributing to an increased frequency of fetal growth restriction (FGR). FGR pregnancies have reduced UtA blood flow partially owing to impaired myometrial artery (MyoA) vasodilation. However, studies show lower rates of HA-associated reductions in fetal growth in Andeans and describe an association between genetic variants predicted to activate the AMP-activated protein kinase (AMPK) pathway and protection against low birthweight. Vascular function studies show that AMPK-dependent vasodilator responses are amplified in UtA from mice exposed to hypoxia during pregnancy and MyoA from pregnant women at HA, while the response is reduced in MyoA from women with FGR pregnancies. The effect of HA (or gestational hypoxia) on placental AMPK activation needs to be clarified, with some studies showing an effect and others not. There is potential to use AMPK activators as therapeutic targets. However, some drugs (i.e. metformin) approved for use in pregnancy complications cause off-target and adverse metabolic effects in offspring, which discourage their use. Future studies are warranted to elucidate the mechanisms underlying the altitude-dependent activation of AMPK in uncomplicated pregnancies and its reduction in FGR to identify possible vascular-specific targets for therapeutic intervention.This article is part of the discussion meeting issue 'Pregnancy at high altitude: the challenge of hypoxia'.

Use of the microfluidic impedance red cell assay in sickle cell disease.

ABSTRACTThis study aimed to compare physiological responses to high‐intensity interval exercise (HIIE) in hypoxia and normoxia across different body mass index (BMI) categories. Twenty‐one males, classified as normal‐weight (NW, n = 9 and BMI: 22.9 ± 2.3 kg · m−2) or overweight/obese (OW, n = 12 and BMI: 27.6 ± 2.0 kg · m−2), completed graded exercise tests (GXT) in normoxia (FiO2 = 20.9%) and hypoxia (FiO2 = 14.0%), followed by three randomised HIIE sessions: hypoxia (HY), normoxia matched to hypoxic relative intensity (NR) and normoxia matched to hypoxic absolute intensity (NA). Blood samples were collected at baseline, immediately post‐HIIE and at 3 and 24 h post‐exercise. Both NW and OW groups had significant reductions in peak heart rate and peak power output in hypoxic versus normoxic GXT (p < 0.05). The NW group showed a greater decline in peak oxygen uptake V˙O2peak under hypoxia compared to OW (Δ = 9.88 ± 5.0 vs. 5.22 ± 3.3 mL · kg · min−1; p < 0.001). OW exhibited increased blood glucose levels post‐hypoxic GXT compared to normoxic conditions (Δ = 0.358 mmol · L−1; p = 0.025). During HIIE sessions, both groups showed similar heart rate, oxygen consumption, carbon dioxide production and respiratory exchange ratio responses. However, blood lactate concentration immediately after normoxic HIIE (NR) was higher in NW compared to OW (p < 0.05). Fasting blood glucose significantly increased immediately after normoxic HIIE in NW and immediately after hypoxic HIIE in OW (p < 0.05). HIIE in normoxia and hypoxia elicits similar physiological responses across BMI categories, though normal‐weight individuals have greater reductions in V˙O2peak and higher lactate responses during normoxic HIIE (NR), whereas overweight/obese individuals exhibit higher glucose increases post‐hypoxic exercise, indicating potential BMI‐specific metabolic benefits. These findings suggest that BMI could influence physiological adaptations in response to high‐intensity exercise in hypoxia, suggesting that this form of exercise could be a beneficial alternative for improving metabolic health, especially in individuals with overweight or obesity.

Introduction: High-intensity resistance training may elevate the risk of musculoskeletal injuries and hinder optimal performance execution. Objective: This study compared the effects of low-load resistance training under blood flow restriction (BFR) and hypoxia (HYP) on body composition, strength, and endurance in untrained male college students. Methodology: Forty-five male college students from Ubon Ratchathani Rajabhat University were purposively sampled and matched into three groups (n = 15): 1) high-load resistance training (HLRT), 2) low-load resistance combined with blood flow restriction training (LLBFR), and 3) low-load resistance combined with hypoxic training (LLHYP). All groups trained three times weekly for five weeks. Results: After five weeks of training, all groups showed significant improvements in fat-free mass, skeletal muscle mass and performance outcome (p &lt; 0.05). The LLHYP group also exhibited significantly reduced skinfold thickness and greater arm circumference (p &lt; 0.05). All groups improved strength and endurance, but LLHYP demonstrated significantly greater endurance than HLRT in both exercises (p = 0.021 and 0.003, respectively) and outperformed LLBFR in the dip machine (p = 0.032). Discussion: Findings support that LLBFR and LLHYP can produce similar strength and body composition outcomes as HLRT over a short term. LLHYP, in particular, showed superior benefits in endurance, possibly due to hypoxia-related physiological adaptations. Conclusions: Low-load resistance training with BFR and HYP effectively enhanced body composition, strength, and endurance of the biceps and triceps. This approach may offer a safer alternative for untrained male students.

Preterm infants are commonly exposed to hyperoxia, which can induce hyperoxia-induced white matter injury (WMI), commonly resulting in cognitive deficits. Existing neonatal rat models of WMI show significant variability. Therefore, this study aimed to develop a reliable rat model of hyperoxia-induced WMI. Two-day-old male newborn rats were randomly assigned to either the hyperoxia (HO) or the normoxia (NO) group. Mice in the HO group were exposed to a high-oxygen-inspired fraction (0.80) for either 24 h, 48 h, 5 d, 7 d, or 10 d, while the NO group was exposed to the standard oxygen-inspired fraction (0.21). Histological examination, immunofluorescence staining, western blot analysis, and transmission electron microscopy were performed to observe myelinogenesis. The Morris water maze test was used to assess cognitive function. The proliferation, migration, differentiation, and apoptosis of oligodendrocytes in the corpus callosum (CC) were evaluated using immunofluorescence. Levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1 (Iba-1), interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were quantified to evaluate oxidative stress and inflammatory responses within the cerebral tissue. Following hyperoxic exposure, demyelination and poor performance in the Morris water maze test were observed in the HO group, notably within the 5 d subgroup (p < 0.05). In addition, compared with the NO group, there were significant oligodendrocyte apoptosis, oxidative stress, and inflammatory responses in ROS, MDA, IL-1β, TNF-α, GFAP, and Iba-1 within the cerebral tissue of the HO group. The numbers of Ki67+/oligodendrocyte transcription factor 2 (Olig2)+ and Vimentin+/Olig2+ cells in the NO and HO groups were not significantly different (p > 0.05). Compared with the NO group, the average fluorescence intensity of Nerve-glia antigen 2 (NG2) and oligodendrocyte-specific marker 4 (O4) in the CC of the HO group increased, whereas the number of cyclic nucleotide phosphodiesterase (CC1) -positive cells significantly decreased (p < 0.05). Hyperoxia causes WMI in neonatal rat brains. Exposure of neonatal rats to 80% oxygen for 5 d induces a reliable animal model of hyperoxia-induced WMI. Aberrant differentiation and apoptosis of oligodendrocytes might be the reason for hyperoxia-induced WMI.

ABSTRACTThis study aimed to evaluate the impact of a four‐week heat and hypoxia training on the fat oxidation capacity of competitive athletes. Eight elite male modern pentathlon athletes completed a four‐week aerobic endurance training program in three environments: normal (CON), high temperature and humidity (HOT), and hypoxia (HYP). Assessments were conducted in both the normal environment and the corresponding special environment before and after training. Gas exchange data were collected during exercise to assess aerobic capacity, and fat oxidation was measured using indirect calorimetry. Fat oxidation kinetics were modeled using the sinusoidal (SIN) mathematical model to determine the maximum fat oxidation (MFO) and the exercise intensity at which it occurred (FATmax). Under normal environment, HOT training had an increase in absolute V̇O2 (238.152 mL/min and p = 0.003), both the HOT (96.062 s and p = 0.006) and HYP (109.917 s and p = 0.002) trainings demonstrated increases in VT2@Time, both the HOT (0.126 g/min and p = 0.015) and HYP (0.157 g/min and p = 0.004) trainings showed increases in MFO, and the HOT training also exhibited an increase in FATmax (5.303 g/min and p = 0.005); both the HOT and HYP trainings showed dilatation of the fat oxidation curve, with the HOT training also displaying dilatation in the fat oxidation curve under heat conditions. Four‐weeks of heat and hypoxia training significantly enhanced athletes' aerobic metabolism and fat oxidation capacity. The benefits of heat training on aerobic metabolism and fat oxidation may exceed those of hypoxia training.

Abstract Rationale. Endothelial nitric oxide (NO) production is a major contributor to systemic vasodilation in hypoxia. By contrast, hypoxic vasoconstriction in the pulmonary vasculature may depend on diminished NO release from pulmonary endothelial cells. NO synthase (NOS) activity is regulated by asymmetric dimethylarginine (ADMA), an endogenous competitive NOS inhibitor. ADMA is metabolized by dimethylarginine dimethylaminohydrolases (DDAH)-1 and -2. We aimed to study the regulation of expression and activity of NOS, DDAH-1 and -2 in primary human coronary (HCAEC) and pulmonary endothelial cells (HPAEC) in normoxia (NX) and hypoxia (HX), and the contribution of alveolar epithelial cells (A549) to this. Methods. HCAEC, HPAEC and A549 cells were cultured in NX (21% O2) and HX (1% O2) for 24h or 72h. eNOS, DDAH1 and DDAH2 mRNA expression was studied by qRT-PCR and protein expression was assessed by Western Blot. Enzymatic activity of DDAH was assessed as the conversion of stable isotope-labeled d7-ADMA to d7-L-citrulline by UPLC-MS/MS. Results. eNOS expression was reduced in HCAEC and HPAEC exposed to HX to a similar degree (HCAEC, 0.64±0.03; HPAEC, 0.73±0.03; both p&amp;lt;0.01 vs. NX). DDAH1 and DDAH2 expression was not significantly changed in HCAEC nor in HPAEC. By contrast, DDAH1 expression was downregulated (to 0.66±0.08, p&amp;lt;0.05) in A549 cells after 24 h of HX, along with higher ADMA release (91.0±26.5 vs. 34.9±6.6 µmol/mg protein; p&amp;lt;0.01). After 72h of HX, we observed a compensatory upregulation of DDAH2 in A549 cells (fold change, 1.85±0.12, p&amp;lt;0.01). This was mirrored by a decrease in DDAH activity (fold change, 0.81±0.14) after 24 h of HX (p &amp;lt; 0.05), which recovered after 72 h of HX in A549 cells (1.03±0.09). ADMA was increased in supernatant of hypoxic A549 cells (91.0±26.5 vs. 34.9±6.6 µmol/mg protein; p&amp;lt;0.01). Conclusions. Despite opposite vascular responses to HX in the systemic and pulmonary circulation, eNOS expression and DDAH-mediated regulation of NOS activity respond similarly in HCAEC and HPAEC. However, DDAH1 is downregulated in pulmonary epithelial cells, which may contribute to intercellular regulation of pulmonary endothelial NO release via elevated ADMA. Thus, downregulation of DDAH1 in hypoxic alveolar epithelial cells may contribute to impaired endothelial NO release in HPAEC during HX and thus aggravate hypoxic pulmonary vasoconstriction. DDAH2 appears to act as a compensatory, counter regulatory enzyme, limiting the impact of DDAH1 downregulation in HX.

Abstract RATIONALE Mesenchymal stromal cell (MSC) therapies have shown promise in preclinical models of pathologies relevant to newborn medicine, such as bronchopulmonary dysplasia (BPD).Previously, we demonstrated that MSC-derived extracellular vesicles (MSC-EVs) improved lung alveolarization and functional outcomes in a murine neonatal hyperoxia (HYRX) model of BPD. In this study, using a double hit model of HYRX followed by allergic asthma induced by house dust mite (HDM) exposure, we investigated the effects of MSC-EVs on long-term immune function and airway hyperresponsiveness (asthma), a known complication of BPD. METHODS Neonatal FVB mice were exposed to HYRX (75% O2) from postnatal day 0 (PN0) to PN7, treated with MSC-EVs between PN2-3, followed by recovery in normoxia (NRMX) until PN14. Subsequently, the mice were sensitized by intranasal instillation of HDM for 5 consecutive days and challenged with HDM on PN28. On PN29, bronchoalveolar lavage fluid (BALF) was collected to evaluate cytokine concentration and cellular composition. Pulmonary function was assessed by challenging mice with increasing doses of methacholine. Additionally, alveolar macrophages (AMs) were isolated from BALF at PN14, stimulated with HDM for 24 hours ex vivo, and their supernatant was added during Th2 polarization. RESULTS Mice exposed to both HYRX and HDM presented with a more severe allergic reaction than HDM alone, characterized by an exacerbated Th2-associated response (Il-4, Il-5, Il-13) along with elevated levels of inflammatory infiltrates in the BALF and markedly higher airway hyper-reactivity. MSC-EV treatment significantly reduced Th2 cytokine production, cellular inflammation, and airway hyperreactivity. Intriguingly, AMs from HYRX animals exhibited higher Il6 levels upon HDM stimulation ex vivo, but this increase was absent in AMs derived from MSC-EV-treated animals. Th2 polarization in the presence of HDM-stimulated AM supernatant resulted in higher levels of Il4 and Il13, but these cytokines were attenuated by MSV-EV treatment, indicating that the in vivo effects of MSC-EVs on AM phenotype are sustained ex vivo. CONCLUSIONS Our results demonstrate that the double-hit model of neonatal HYRX and HDM leads to a significantly higher inflammatory response and airway disease compared to individual exposures. Importantly, MSC-EV-treated mice normalized the oxygen-disrupted lung development by modulating the functionality of AMs resulting in diminished exacerbation of HDM-induced inflammation and airway hyperresponsiveness. These findings suggest that MSC-EV therapy is a promising strategy for mitigating the long-term pulmonary complications of infants with BPD.

Abstract RATIONALE: Lung matrix remodeling by activated fibroblasts is a key pathomechanism of bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF). Epigenetic modifiers could contribute to the onset and progression of lung fibrosis. We recently identified Krüppel-like factor 4 (Klf4) as a key transcription factor in fibroblast cellular activity through an EP300-related interactome. Hence, we now studied if the Klf4-Ep300 axis is a regulator of the acetylome and of chromatin remodeling in fibroblasts in BPD and IPF, favoring thereby lung fibrosis.METHODS: (i) a neonatal murine exposed to hyperoxia (HYX) or normoxia (NOX) until postnatal day (P)14 or P28, (ii) transgenic mice with an ACTA2+ cell-specific deletion of Klf4, (iii) primary neonatal murine and human lung fibroblasts from lungs with IPF and control, and (iv) lungs from patients with BPD or IPF vs control. RESULTS: HYX caused fibrotic lung remodeling in neonatal hyperoxia-exposed mice that was associated with an active TGFβ signaling and reduced Klf4 abundance in proliferative ACTA2+ cells. These findings were related to changes in global and posttranslational protein acetylation as well as to an activation of EP300 in ACTA2+ cells in vivo and in cultured lung fibroblasts, that results in an inactivation of FoxO1. Transgenic mice with ACTA2+ cell-specific ablation of Klf4 exhibited higher matrix remodeling with increased number ACTA2+ cells as well as activation of EP300 and altered histone acetylation. Next, we found Klf4 to regulate proliferation, migration and differentiation in primary neonatal murine and in human lung fibroblasts. Chip-Seq, biotinylation identication assay (BioID), and EP300 inhibition supported a functional role of Klf4 as a key regulator of the global acetylome and FoxO1 acetylation. In addition, we performed ATAC-seq of murine lung fibroblasts from transgenic mice with ACTA2+ cell-specific ablation of Klf4 and from human donor fibroblasts with Klf4 knockdown. Deep epigenetic profiling uncovered a shifted transcription factor footprint favoring FoxO signaling and fibrosis following the loss of Klf4, along with a marked converging epigenetic profile with human IPF-fibroblasts. Finally, human lungs with BPD exhibited significantly reduced abundance of Klf4+ ACTA2+ and an increase in ACTA2+ cells. Similarly, fibrotic foci of IPF lungs showed loss of Klf4. Conversely, Klf4 overexpression exhibited an anti-proliferative and pro-apoptotic effect in IPF lung fibroblasts.CONCLUSION: Our study reveals a Klf4-Ep300 axis as a novel epigenetic modifier of chromatin architecture in lung fibroblasts through modulation of the acetylome, favouring thereby fibrosis and offering a possible therapeutic target for severe BPD and IPF.

Abstract Cigarette smoke (CS) is a significant risk factor for developing acute respiratory distress syndrome (ARDS), but the cellular and molecular mechanisms linking smoking to ARDS susceptibility remain unclear. Our goal was to improve our understanding of these mechanisms. To address this, we established a mouse model comparing long-term CS exposure to non-smoking controls, examining responses to influenza infection and hyperoxia-induced lung injury. The mice were divided into six groups (n=1 per group): control (CON), cigarette smoke (CS), hyperoxia (HYP), influenza infection (FLU), cigarette smoke plus hyperoxia (CS+HYP), and cigarette smoke plus influenza infection (CS+FLU). Single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) were performed on lung tissues. Quality control analysis using Seurat (v5.1.0) and Signac (v1.13.1) retained 78,402 cells from scRNA-seq and 84,144 cells from scATAC-seq, with 32,305 matched cells identified across both datasets. Differential gene expression analysis revealed significant smoking-associated alterations in cellular responses to influenza infection and hyperoxia exposure. Pathway enrichment indicated heightened immune responses, inflammatory signaling, and cellular survival pathways in smoking-exposed animals. Integration of scRNA-seq and scATAC-seq identified key transcription factors (TFs), including those involved in immune regulation, tissue repair, and chromatin remodeling mediating these responses. Overall, this study underscores the role of chronic cigarette smoke exposure in exacerbating pathways critical to ARDS pathogenesis, providing potential targets for therapeutic intervention.

The hypoxic microenvironment of tumor cells is closely related to the progression of ovarian cancer (OV). Hypoxia (HY)-related matrix-remodeling associated 5 (MXRA5) was expressed at elevated levels in many tumors, but research on the impact of MXRA5 in OV remains limited. This study aims to explore the role of MXRA5 in regulating cellular HY in OV. The MXRA5 expression and its clinical significance in OV were evaluated using GEPIA2, Kaplan-Meier plotter databases, and immunohistochemistry assay. OV cells were treated with normoxia and HY conditions. The siRNAs were designed to knock down the MXRA5 expression in hypoxic cells. The cellular capacities were detected by CCK-8 assay, EdU assay, Transwel assay, and TUNEL assay, each method targeting a different aspect of cellular behavior. The MXRA5 level was increased in OV and associated with the progression free survival and overall survival of OV patients. The proliferation and invasion abilities of OV cells were promoted, while apoptosis capacities were inhibited in hypoxic cells. After the knockdown of MXRA5 in hypoxic cells, the proliferative capacities and invasive abilities of the cells were reduced, and the apoptosis capacities were enhanced. Moreover, mechanistically, HIF-1α is a key transcription factor in response to HY that binds to the MXRA5 promoter. MXRA5 expression was induced by HY and had prognostic performance in OV. Knockdown of MXRA5 can inhibit proliferation and invasion in OV cells caused by HIF-1α, revealing that MXRA5 is one potential targets for tumor HY regulation in OV.