TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Mitophagy mediated by HIF-1α/FUNDC1 signaling in tubular cells protects against renal ischemia/reperfusion injury.

Acute kidney injury (AKI) is associated with a high mortality rate. Pathologically, renal ischemia/reperfusion injury (RIRI) is one of the primary causes of AKI, and hypoxia-inducible factor (HIF)-1α may play a defensive role in RIRI. This study assessed the role of hypoxia-inducible factor 1α (HIF-1α)-mediated mitophagy in protection against RIRI in vitro and in vivo. The human tubular cell line HK-2 was used to assess hypoxia/reoxygenation (H/R)-induced mitophagy through different in vitro assays, including western blotting, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and reactive oxygen species (ROS) measurement. Additionally, a rat RIRI model was established for evaluation by renal histopathology, renal Doppler ultrasound, and transmission electron microscopy to confirm the in vitro data. The selective HIF-1α inhibitor LW6 reduced H/R-induced mitophagy but increased H/R-induced apoptosis and ROS production. Moreover, H/R treatment enhanced expression of the FUN14 domain-containing 1 (FUNDC1) protein. Additionally, FUNDC1 overexpression reversed the effects of LW6 on the altered expression of light chain 3 (LC3) BII and voltage-dependent anion channels as well as blocked the effects of HIF-1α inhibition in cells. Pretreatment of the rat RIRI model with roxadustat, a novel oral HIF-1α inhibitor, led to decreased renal injury and apoptosis in vivo. In conclusion, the HIF-1α/FUNDC1 signaling pathway mediates H/R-promoted renal tubular cell mitophagy, whereas inhibition of this signaling pathway protects cells from mitophagy, thus aggravating apoptosis, and ROS production. Accordingly, roxadustat may protect against RIRI-related AKI.

Xinnaotongluo liquid protects H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3.

Xinnaotongluo liquid has been used to improve the clinical symptoms of patients with myocardial infarction. However, the molecular mechanism of Xinnaotongluo liquid is not completely understood. H9c2 cells exposed to hypoxia/reoxygenation (H/R) was used to simulate damage to cardiomyocytes in myocardial infarction in vitro. The biological indicators of H9c2 cells were measured by cell counting kit-8, enzyme linked immunoabsorbent assay, and western blot assay. In H/R-induced H9c2 cells, a markedly reduced murine double minute 2 (MDM2) was observed. However, the addition of Xinnaotongluo liquid increased MDM2 expression in H/R-induced H9c2 cells. And MDM2 overexpression strengthened the beneficial effects of Xinnaotongluo liquid on H9c2 cells from the perspective of alleviating oxidative damage, cellular inflammation, apoptosis and ferroptosis of H/R-induced H9c2 cells. Moreover, MDM2 overexpression reduced the protein expression of p53 and Six-Transmembrane Epithelial Antigen of Prostate 3 (STEAP3). Whereas, STEAP3 overexpression hindered the function of MDM2-overexpression in H/R-induced H9c2 cells. Our results insinuated that Xinnaotongluo liquid could protect H9c2 cells from H/R-induced damage by regulating MDM2/STEAP3, which provide a potential theoretical basis for further explaining the working mechanism of Xinnaotongluo liquid.

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

BACKGROUND: Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. METHOD: The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. RESULTS: HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. CONCLUSION: HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Metabolomics analysis revealed the neuroprotective role of 2-phosphoglyceric acid in hypoxic-ischemic brain damage through GPX4/ACSL4 axis regulation.

Hypoxic-ischemic brain damage (HIBD) is a cerebral injury resulting from the combination of ischemia and hypoxia in neonatal brain tissue. Presently, there exists no efficacious remedy for HIBD. A mounting body of evidence indicates that dynamic metabolites formed during metabolic procedures assume a vital role in neuronal maturation and recuperation. However, it remains unclear whether any endogenous metabolites are involved in the pathogenesis of HIBD. Here, an untargeted metabolomics analysis was conducted by gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry (GC/LC-MS) in OGD/R (oxygen-glucose deprivation/reoxygenation)-induced HT-22 cells. We observed that ferroptosis signaling plays an essential role in HI-induced neuronal injury. Interestingly, we also found that the differentially expressed metabolite, 2-phosphoglyceric acid, significantly improved the neuronal cell survival of OGD/R HT-22 cells by inhibiting ferroptosis. Moreover, 2-phosphoglyceric acid effectively rescued the cell activity of HT-22 cells treated with the ferroptosis inducer RSL-3. Furthermore, 2-phosphoglyceric acid alleviated cerebral infarction and reduced HIBD-induced neuronal cell loss of the central nervous system in neonatal rats by regulating GPX4 expression. Taken together, we found that 2-phosphoglyceric acid, which was downregulated in HT-22 cells induced by OGD/R, exerted neuronal protective effects on OGD/R-treated HT-22 cells and HIBD-induced neonatal rats by inhibiting hypoxic-ischemic-induced ferroptosis through the regulation of the GPX4/ACSL4 axis.

Two hexokinases of the shrimp Penaeus (Litopenaeus) vannamei are differentially expressed during oxygen limited conditions.

The white shrimp Penaeus (Litopenaeus) vannamei is the most cultivated shrimp worldwide. Compared to other shrimp species, it has higher resistance to adverse conditions. During hypoxia, the shrimp reduces oxygen consumption and adjusts energy metabolism via anaerobic glycolysis, among other strategies. Hexokinase (HK) is the first enzyme of glycolysis and a key regulation point. In mammals and other vertebrates, there are several tissue-specific HK isoforms with differences in expression and enzyme activity. In contrast, crustacean HKs have been relatively little studied. We studied the P. vannamei HK isoforms during hypoxia and reoxygenation. We cloned two HK1 sequences named HK1-long (1455 bp) and HK1-short (1302 bp), and one HK2 (1344 bp). In normoxia, total HK1 expression is higher in hepatopancreas, while HK2 is higher in gills. Severe hypoxia (1 mg/L of DO) after 12 h exposure and 1 h of reoxygenation increased HK1 expression in both organs, but HK2 expression changed differentially. In hepatopancreas, HK2 expression increased in 6 and 12 h of hypoxia but diminished to normoxia levels after reoxygenation. In gills, HK2 expression decreased after 12 h of hypoxia. HK activity increased in hepatopancreas after 12 h hypoxia, opposite to gills. These results indicate that shrimp HK isoforms respond to hypoxia and reoxygenation in a tissue-specific manner. Intracellular glucose levels did not change in any case, showing the shrimp ability to maintain glucose homeostasis during hypoxia.

Fructose improves titanium dioxide nanoparticles induced alterations in developmental competence of mouse oocytes.

AIMS: We investigated the effects of intraperitoneal injections of titanium dioxide nanoparticles (TiO2 NPs, 100 mg/kg) for 5 consecutive days on the developmental competence of murine oocytes. Furthermore, study the effects of TiO2 NPs on antioxidant and oxidative stress biomarkers, as well as their effects on expression of apoptotic and hypoxia inducing factor-1α (HIF1A) protein translation. Moreover, the possible ameliorating effects of intraperitoneal injections of fructose (2.75 mM/ml) was examined. MATERIALS AND METHODS: Thirty sexually mature (8-12 weeks old; ~ 25 g body weight) female mice were used for the current study. The female mice were assigned randomly to three treatment groups: Group1 (G1) mice were injected intraperitoneal (ip) with deionized water for 5 consecutive days; Group 2 (G2) mice were injected ip with TiO2 NPs (100 mg/kg BW) for 5 consecutive days; Group 3 (G3) mice were injected ip with TiO2 NPs (100 mg/kg BW + fructose (2.75 mM) for 5 consecutive days. RESULTS: Nano-titanium significantly decreased expression of GSH, GPx, and NO, expression of MDA and TAC increased. The rates of MI, MII, GVBD and degenerated oocytes were significantly less for nano-titanium treated mice, but the rate of activated oocytes was significantly greater than those in control oocytes. TiO2 NPs significantly increased expression of apoptotic genes (BAX, Caspase 3 and P53) and HIF1A. Intraperitoneal injection of fructose (2.75 mM/kg) significantly alleviated the detrimental effects of TiO2 NPs. Transmission electron microscopy indicated that fructose mitigated adverse effects of TiO2 NPs to alter the cell surface of murine oocytes. CONCLUSION: Results of this study suggest that the i/p infusion of fructose for consecutive 5 days enhances development of murine oocytes and decreases toxic effects of TiO2 NPs through positive effects on oxidative and antioxidant biomarkers in cumulus-oocyte complexes and effects to inhibit TiO2-induced increases in expression of apoptotic and hypoxia inducing factors.

Hypoxia treatment and resistance training alters microRNA profiling in rats skeletal muscle.

MicroRNAs (miRNAs) may play a crucial regulatory role in the process of muscle atrophy induced by high-altitude hypoxia and its amelioration through resistance training. However, research in this aspect is still lacking. Therefore, this study aimed to employ miRNA microarray analysis to investigate the expression profile of miRNAs in skeletal muscle from an animal model of hypoxia-induced muscle atrophy and resistance training aimed at mitigating muscle atrophy. The study utilized a simulated hypoxic environment (oxygen concentration at 11.2%) to induce muscle atrophy and established a rat model of resistance training using ladder climbing, with a total intervention period of 4 weeks. The miRNA expression profile revealed 9 differentially expressed miRNAs influenced by hypoxia (e.g., miR-341, miR-32-5p, miR-465-5p) and 14 differentially expressed miRNAs influenced by resistance training under hypoxic conditions (e.g., miR-338-5p, miR-203a-3p, miR-92b-3p) (∣log2(FC)∣ ≥ 1.5, p < 0.05). The differentially expressed miRNAs were found to target genes involved in muscle protein synthesis and degradation (such as Utrn, mdm2, eIF4E), biological processes (such as negative regulation of transcription from RNA polymerase II promoter, regulation of transcription, DNA-dependent), and signaling pathways (such as Wnt signaling pathway, MAPK signaling pathway, ubiquitin-mediated proteolysis, mTOR signaling pathway). This study provides a foundation for understanding and further exploring the molecular mechanisms underlying hypoxia-induced rats muscle atrophy and the mitigation of atrophy through resistance training.

Crosstalk between hypoxia-inducible factor-1α and short-chain fatty acids in inflammatory bowel disease: key clues toward unraveling the mystery.

The human intestinal tract constitutes a complex ecosystem, made up of countless gut microbiota, metabolites, and immune cells, with hypoxia being a fundamental environmental characteristic of this ecology. Under normal physiological conditions, a delicate balance exists among these complex "residents", with disruptions potentially leading to inflammatory bowel disease (IBD). The core pathology of IBD features a disrupted intestinal epithelial barrier, alongside evident immune and microecological disturbances. Central to these interconnected networks is hypoxia-inducible factor-1α (HIF-1α), which is a key regulator in gut cells for adapting to hypoxic conditions and maintaining gut homeostasis. Short-chain fatty acids (SCFAs), as pivotal gut metabolites, serve as vital mediators between the host and microbiota, and significantly influence intestinal ecosystem. Recent years have seen a surge in research on the roles and therapeutic potential of HIF-1α and SCFAs in IBD independently, yet reviews on HIF-1α-mediated SCFAs regulation of IBD under hypoxic conditions are scarce. This article summarizes evidence of the interplay and regulatory relationship between SCFAs and HIF-1α in IBD, pivotal for elucidating the disease's pathogenesis and offering promising therapeutic strategies.

From Vessels to Neurons-The Role of Hypoxia Pathway Proteins in Embryonic Neurogenesis.

Embryonic neurogenesis can be defined as a period of prenatal development during which divisions of neural stem and progenitor cells give rise to neurons. In the central nervous system of most mammals, including humans, the majority of neocortical neurogenesis occurs before birth. It is a highly spatiotemporally organized process whose perturbations lead to cortical malformations and dysfunctions underlying neurological and psychiatric pathologies, and in which oxygen availability plays a critical role. In case of deprived oxygen conditions, known as hypoxia, the hypoxia-inducible factor (HIF) signaling pathway is activated, resulting in the selective expression of a group of genes that regulate homeostatic adaptations, including cell differentiation and survival, metabolism and angiogenesis. While a physiological degree of hypoxia is essential for proper brain development, imbalanced oxygen levels can adversely affect this process, as observed in common obstetrical pathologies such as prematurity. This review comprehensively explores and discusses the current body of knowledge regarding the role of hypoxia and the HIF pathway in embryonic neurogenesis of the mammalian cortex. Additionally, it highlights existing gaps in our understanding, presents unanswered questions, and provides avenues for future research.

OPN silencing reduces hypoxic pulmonary hypertension via PI3K-AKT-induced protective autophagy.

Hypoxic pulmonary hypertension (HPH) is a pulmonary vascular disease primarily characterized by progressive pulmonary vascular remodeling in a hypoxic environment, posing a significant clinical challenge. Leveraging data from the Gene Expression Omnibus (GEO) and human autophagy-specific databases, osteopontin (OPN) emerged as a differentially expressed gene, upregulated in cardiovascular diseases such as pulmonary arterial hypertension (PAH). Despite this association, the precise mechanism by which OPN regulates autophagy in HPH remains unclear, prompting the focus of this study. Through biosignature analysis, we observed significant alterations in the PI3K-AKT signaling pathway in PAH-associated autophagy. Subsequently, we utilized an animal model of OPNfl/fl-TAGLN-Cre mice and PASMCs with OPN shRNA to validate these findings. Our results revealed right ventricular hypertrophy and elevated mean pulmonary arterial pressure (mPAP) in hypoxic pulmonary hypertension model mice. Notably, these effects were attenuated in conditionally deleted OPN-knockout mice or OPN-silenced hypoxic PASMCs. Furthermore, hypoxic PASMCs with OPN shRNA exhibited increased autophagy compared to those in hypoxia alone. Consistent findings from in vivo and in vitro experiments indicated that OPN inhibition during hypoxia reduced PI3K expression while increasing LC3B and Beclin1 expression. Similarly, PASMCs exposed to hypoxia and PI3K inhibitors had higher expression levels of LC3B and Beclin1 and suppressed AKT expression. Based on these findings, our study suggests that OPNfl/fl-TAGLN-Cre effectively alleviates HPH, potentially through OPN-mediated inhibition of autophagy, thereby promoting PASMCs proliferation via the PI3K-AKT signaling pathway. Consequently, OPN emerges as a novel therapeutic target for HPH.

Shen Shuai II recipe improves renal hypoxia to attenuate renal injury in 5/6 renal ablation/infarction rats and effect evaluation using blood oxygenation level-dependent functional magnetic resonance imaging.

Background: Renal hypoxia plays a key role in the progression of chronic kidney disease (CKD). Shen Shuai II Recipe (SSR) has shown good results in the treatment of CKD as a common herbal formula. This study aimed to explore the effect of SSR on renal hypoxia and injury in CKD rats. Methods: Twenty-five Wistar rats underwent 5/6 renal ablation/infarction (A/I) surgery were randomly divided into three groups: 5/6 (A/I), 5/6 (A/I) + losartan (LOS), and 5/6 (A/I) + SSR groups. Another eight normal rats were used as the Sham group. After 8-week corresponding interventions, blood oxygenation level-dependent functional magnetic resonance imaging (BOLD-fMRI) was performed to evaluate renal oxygenation in all rats, and biochemical indicators were used to measure kidney and liver function, hemoglobin, and proteinuria. The expression of fibrosis and hypoxia-related proteins was analyzed using immunoblotting examination. Results: Renal oxygenation, evaluated by BOLD-fMRI as cortical and medullary T2* values (COT2* and MET2*), was decreased in 5/6 (A/I) rats, but increased after SSR treatment. SSR also downregulated the expression of hypoxia-inducible factor-1α (HIF-1α) in 5/6 (A/I) kidneys. With the improvement of renal hypoxia, renal function and fibrosis were improved in 5/6 (A/I) rats, accompanied by reduced proteinuria. Furthermore, the COT2* and MET2* were significantly positively correlated with the levels of creatinine clearance rate (Ccr) and hemoglobin, but negatively associated with the levels of serum creatinine (SCr), blood urea nitrogen (BUN), serum cystatin C (CysC), serum uric acid (UA), 24-h urinary protein (24-h Upr), and urinary albumin:creatinine ratio (UACR). Conclusion: The degree of renal oxygenation reduction is correlated with the severity of renal injury in CKD. SSR can improve renal hypoxia to attenuate renal injury in 5/6 (A/I) rats of CKD.

Alfaxalone is an effective anesthetic for the electrophysiological study of anoxia-tolerance mechanisms in western painted turtle pyramidal neurons.

Anoxia in the mammalian brain leads to hyper-excitability and cell death; however, this cascade of events does not occur in the anoxia-tolerant brain of the western painted turtle, Chrysemys picta belli. The painted turtle has become an important anoxia-tolerant model to study brain, heart, and liver function in the absence of oxygen, but being anoxia-tolerant likely means that decapitation alone is not a suitable method of euthanasia. Many anesthetics have long-term effects on ion channels and are not appropriate for same day experimentation. Using whole-cell electrophysiological techniques, we examine the effects of the anesthetic, Alfaxalone, on pyramidal cell action potential amplitude, threshold, rise and decay time, width, frequency, whole cell conductance, and evoked GABAA receptors currents to determine if any of these characteristics are altered with the use of Alfaxalone for animal sedation. We find that Alfaxalone has no long-term impact on action potential parameters or whole-cell conductance. When acutely applied to naïve tissue, Alfaxalone did lengthen GABAA receptor current decay rates by 1.5-fold. Following whole-animal sedation with Alfaxalone, evoked whole cell GABAA receptor current decay rates displayed an increasing trend with 1 and 2 hours after brain sheet preparation, but showed no significant change after a 3-hour washout period. Therefore, we conclude that Alfaxalone is a suitable anesthetic for same day use in electrophysiological studies in western painted turtle brain tissue.

PPARγ alleviates preeclampsia development by regulating lipid metabolism and ferroptosis.

The study aims to explore the effect of PPARγ signaling on ferroptosis and preeclampsia (PE) development. Serum and placental tissue are collected from healthy subjects and PE patients. The PPARγ and Nrf2 decreases in the PE. Rosiglitazone intervention reverses hypoxia-induced trophoblast ferroptosis and decreases lipid synthesis by regulating Nfr2 and SREBP1. Compared to the Hypoxia group, the migratory and invasive abilities enhance after rosiglitazone and ferr1 treatment. Rosiglitazone reduces the effect of hypoxia and erastin. The si-Nrf2 treatment attenuats the effects of rosiglitazone on proliferation, migration, and invasion. The si-Nrf2 does not affect SREBP1 expression. PPARγ agonists alleviates ferroptosis in the placenta of the PE rats. The study confirms that PPARγ signaling and ferroptosis-related indicators were dysregulated in PE. PPARγ/Nrf2 signaling affects ferroptosis by regulating lipid oxidation rather than SREBP1-mediated lipid synthesis. In conclusion, our study find that PPARγ can alleviate PE development by regulating lipid oxidation and ferroptosis.

Selective degradation of PL2L60 by metabolic stresses­induced autophagy suppresses multi­cancer growth.

It has been reported that PL2L60 proteins, a product of PIWIL2 gene which might be activated by an intragenic promoter, could mediate a common pathway specifically for tumorigenesis. In the present study, it was further identified by using western blot assay that the PL2L60 proteins could be degraded in cancer cells through a mechanism of selective autophagy in response to oxidative stress. The PL2L60 was downregulated in various types of cancer cells under the hypoxic condition independently of HIF­1α, resulting in apoptosis of cancer cells. Inhibition of autophagy by small interfering RNA targeting of either Beclin­1 (BECN1) or Atg5 resulted in restoration of PL2L60 expression in hypoxic cancer cell. The hypoxic degradation of PL2L60 was also blocked by the attenuation of the autophagosome membrane protein Atg8/microtubule­associated protein 1 light chain 3 (LC3) or autophagy cargo protein p62 expression. Surprisingly, Immunofluorescence analysis demonstrated that LC3 could be directly bound to PL2L60 and was required for the transport of PL2L60 from the nucleus to the cytoplasm for lysosomal flux under basal or activated autophagy in cancer cells. Moreover, flow cytometric analysis displayed that knocking down of PL2L60 mRNA but not PIWIL2 mRNA effectively inhibited cancer cell proliferation and promoted apoptosis of cancer cells. The similar results were obtained from in vivo tumorigenic experiment, in which PL2L60 downregulation in necroptosis areas was confirmed by immunohistochemistry. These results suggested that various cancer could be suppressed by promoting autophagy. The present study revealed a key role of autophagic degradation of PL2L60 in hypoxia­induced cancer cell death, which could be used as a novel therapeutic target of cancer.

Chronic intermittent hypoxia reveals role of the Postinspiratory Complex in the mediation of normal swallow production.

Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder that results in multiple bouts of intermittent hypoxia. OSA has many neurological and systemic comorbidities, including dysphagia, or disordered swallow, and discoordination with breathing. However, the mechanism in which chronic intermittent hypoxia (CIH) causes dysphagia is unknown. Recently, we showed the postinspiratory complex (PiCo) acts as an interface between the swallow pattern generator (SPG) and the inspiratory rhythm generator, the preBötzinger complex, to regulate proper swallow-breathing coordination (Huff et al., 2023). PiCo is characterized by interneurons co-expressing transporters for glutamate (Vglut2) and acetylcholine (ChAT). Here we show that optogenetic stimulation of ChATcre:Ai32, Vglut2cre:Ai32, and ChATcre:Vglut2FlpO:ChR2 mice exposed to CIH does not alter swallow-breathing coordination, but unexpectedly disrupts swallow behavior via triggering variable swallow motor patterns. This suggests that glutamatergic-cholinergic neurons in PiCo are not only critical for the regulation of swallow-breathing coordination, but also play an important role in the modulation of swallow motor patterning. Our study also suggests that swallow disruption, as seen in OSA, involves central nervous mechanisms interfering with swallow motor patterning and laryngeal activation. These findings are crucial for understanding the mechanisms underlying dysphagia, both in OSA and other breathing and neurological disorders.

Repeated sprint training in hypoxia induces specific skeletal muscle adaptations through S100A protein signaling.

Athletes increasingly engage in repeated sprint training consisting in repeated short all-out efforts interspersed by short recoveries. When performed in hypoxia (RSH), it may lead to greater training effects than in normoxia (RSN); however, the underlying molecular mechanisms remain unclear. This study aimed at elucidating the effects of RSH on skeletal muscle metabolic adaptations as compared to RSN. Sixteen healthy young men performed nine repeated sprint training sessions in either normoxia (FIO2 = 0.209, RSN, n = 7) or normobaric hypoxia (FIO2 = 0.136, RSH, n = 9). Before and after the training period, exercise performance was assessed by using repeated sprint ability (RSA) and Wingate tests. Vastus lateralis muscle biopsies were performed to investigate muscle metabolic adaptations using proteomics combined with western blot analysis. Similar improvements were observed in RSA and Wingate tests in both RSN and RSH groups. At the muscle level, RSN and RSH reduced oxidative phosphorylation protein content but triggered an increase in mitochondrial biogenesis proteins. Proteomics showed an increase in several S100A family proteins in the RSH group, among which S100A13 most strongly. We confirmed a significant increase in S100A13 protein by western blot in RSH, which was associated with increased Akt phosphorylation and its downstream targets regulating protein synthesis. Altogether our data indicate that RSH may activate an S100A/Akt pathway to trigger specific adaptations as compared to RSN.

Exosome-coated oxygen nanobubble-laden hydrogel augments intracellular delivery of exosomes for enhanced wound healing.

Wound healing is an obvious clinical concern that can be hindered by inadequate angiogenesis, inflammation, and chronic hypoxia. While exosomes derived from adipose tissue-derived stem cells have shown promise in accelerating healing by carrying therapeutic growth factors and microRNAs, intracellular cargo delivery is compromised in hypoxic tissues due to activated hypoxia-induced endocytic recycling. To address this challenge, we have developed a strategy to coat oxygen nanobubbles with exosomes and incorporate them into a polyvinyl alcohol/gelatin hybrid hydrogel. This approach not only alleviates wound hypoxia but also offers an efficient means of delivering exosome-coated nanoparticles in hypoxic conditions. The self-healing properties of the hydrogel, along with its component, gelatin, aids in hemostasis, while its crosslinking bonds facilitate hydrogen peroxide decomposition, to ameliorate wound inflammation. Here, we show the potential of this multifunctional hydrogel for enhanced healing, promoting angiogenesis, facilitating exosome delivery, mitigating hypoxia, and inhibiting inflammation in a male rat full-thickness wound model.

Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals.

BACKGROUND: Elephant seals exhibit extreme hypoxemic tolerance derived from repetitive hypoxia/reoxygenation episodes they experience during diving bouts. Real-time assessment of the molecular changes underlying protection against hypoxic injury in seals remains restricted by their at-sea inaccessibility. Hence, we developed a proliferative arterial endothelial cell culture model from elephant seals and used RNA-seq, functional assays, and confocal microscopy to assess the molecular response to prolonged hypoxia. RESULTS: Seal and human endothelial cells exposed to 1% O2 for up to 6 h respond differently to acute and prolonged hypoxia. Seal cells decouple stabilization of the hypoxia-sensitive transcriptional regulator HIF-1α from angiogenic signaling. Rapid upregulation of genes involved in glutathione (GSH) metabolism supports the maintenance of GSH pools, and intracellular succinate increases in seal but not human cells. High maximal and spare respiratory capacity in seal cells after hypoxia exposure occurs in concert with increasing mitochondrial branch length and independent from major changes in extracellular acidification rate, suggesting that seal cells recover oxidative metabolism without significant glycolytic dependency after hypoxia exposure. CONCLUSIONS: We found that the glutathione antioxidant system is upregulated in seal endothelial cells during hypoxia, while this system remains static in comparable human cells. Furthermore, we found that in contrast to human cells, hypoxia exposure rapidly activates HIF-1 in seal cells, but this response is decoupled from the canonical angiogenesis pathway. These results highlight the unique mechanisms that confer extraordinary tolerance to limited oxygen availability in a champion diving mammal.

Expression of STAT3 and hypoxia markers in long-term surviving malignant glioma patients.

BACKGROUND: Glioblastoma is a malignant and aggressive type of central nevous system malignancy characterized by many distinct biological features including extensive hypoxia. Hypoxia in glioblatoma associates with complex signaling patterns including activation of several pathways such as MAPK, PI3K-AKT/mTOR and IL-6/JAK/STAT3 with the master regulator HIF-1, which in turn drive particular tumor behaviors determining, in the end, treatment outcomes and patients fate. Thus, the present study was designed to investigate the expression of selected hypoxia related factors including STAT3 in a small set of long-term surviving glioma patients. METHODS: The expression of selected hypoxia related factors including STAT3 was evaluated in a time series of formalin fixed paraffin embedded and cryopreserved glioma samples from repeatedly resected patients. In addition, comparative studies were also conducted on primary glioma cells derived from original patient samples, stabilized glioma cell lines and tumor-xenograft mice model. Obtained data were correlated with clinical findings too. RESULTS: Glioblastoma samples of the analyzed patients displayed heterogeneity in the expression of hypoxia- related and EMT markers with most interesting trend being observed in pSTAT3. This heterogeneity was subsequently confirmed in other employed models (primocultures derived from glioblastoma tissue resections, cryopreserved tumor specimens, stabilized glioblastoma cell line in vitro and in vivo) and concerned, in particular, STAT3 expression which remained stable. In addition, subsequent studies on the role of STAT3 in the context of glioblastoma hypoxia demonstrated opposing effects of its deletion on cell viability as well as the expression of hypoxia and EMT markers. CONCLUSIONS: Our results suport the importance of STAT3 expression and activity in the context of hypoxia in malignant glioblastoma long-term surviving glioma patients while emphasizing heterogeneity of biological outcomes in varying employed tumor models.