Impact of Zinc on Oxidative Signaling Pathways in the Development of Pulmonary Vasoconstriction Induced by Hypobaric Hypoxia

Hypobaric hypoxia is a condition that occurs at high altitudes (>2500 m) where the partial pressure of gases, particularly oxygen (PO₂), decreases. This condition triggers several physiological and molecular responses. One of the principal responses is pulmonary vascular contraction, which seeks to optimize gas exchange under this condition, known as hypoxic pulmonary vasoconstriction (HPV); however, when this physiological response is exacerbated, it contributes to the development of high-altitude pulmonary hypertension (HAPH). Increased levels of zinc (Zn²⁺) and oxidative stress (known as the “ROS hypothesis”) have been demonstrated in the vasoconstriction process. Therefore, the aim of this review is to determine the relationship between molecular pathways associated with altered Zn²⁺ levels and oxidative stress in HPV in hypobaric hypoxic conditions. The results indicate an increased level of Zn²⁺, which is related to increasing mitochondrial ROS (mtROS), alterations in nitric oxide (NO), metallothionein (MT), zinc-regulated, iron-regulated transporter-like protein (ZIP), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-induced protein kinase C epsilon (PKCε) activation in the development of HPV. In conclusion, there is an association between elevated Zn²⁺ levels and oxidative stress in HPV under different models of hypoxia, which contribute to understanding the molecular mechanism involved in HPV to prevent the development of HAPH.     

From Tissue Physoxia to Cancer Hypoxia,Cost-Effective Methods to Study Tissue-Specific O2 Levels in Cellular Biology

The human body is endowed with an extraordinary ability to maintain different oxygen levels in various tissues and organs. The maintenance of physiological levels of oxygen is known as physoxia. The development of hypoxic conditions plays an important role in the biology of several pathologies, including cancer. In vitro studies using normal and neoplastic cells require that culture conditions be carried out under appropriate oxygen levels, either physoxic or hypoxic conditions. Such requirements are difficult to widely implement in laboratory practice, mainly due to the high costs of specialized equipment. In this work, we present and characterize a cost-effective method to culture cells under a range of oxygen levels using deoxidizing pouches. Our results show that physoxic and hypoxic levels using deoxidizing absorbers can be achieved either by implementing a gradual change in oxygen levels or by a regimen of acute depletion of oxygen. This approach triggers the activation of an epithelial-mesenchymal transition in cancer cells while stimulating the expression of HIF-1α. Culturing cancer cells with deoxidizing agent pouches revealed PI3K oncogenic pathway exacerbations compared to tumor cells growing under atmospheric levels of oxygen. Similar to the PI3K signaling disturbance, we also observed augmented oxidative stress and superoxide levels and increased cell cycle arrest. Most interestingly, the culture of cancer cells under hypoxia resulted in the accumulation of cancer stem cells in a time-dependent manner. Overall, we present an attractive, cost-effective method of culturing cells under appropriate physoxic or hypoxic conditions that is easily implementable in any wet laboratory equipped with cell culture tools.     

Proteomic Analysis Reveals That Mitochondria Dominate the Hippocampal Hypoxic Response in Mice

Hypoxic stress occurs in various physiological and pathological states, such as aging, disease, or high-altitude exposure, all of which pose a challenge to many organs in the body, necessitating adaptation. However, the exact mechanisms by which hypoxia affects advanced brain function (learning and memory skills in particular) remain unclear. In this study, we investigated the effects of hypoxic stress on hippocampal function. Specifically, we studied the effects of the dysfunction of mitochondrial oxidative phosphorylation using global proteomics. First, we found that hypoxic stress impaired cognitive and motor abilities, whereas it caused no substantial changes in the brain morphology or structure of mice. Second, bioinformatics analysis indicated that hypoxia affected the expression of 516 proteins, of which 71.1% were upregulated and 28.5% were downregulated. We demonstrated that mitochondrial function was altered and manifested as a decrease in NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 expression, accompanied by increased reactive oxygen species generation, resulting in further neuronal injury. These results may provide some new insights into how hypoxic stress alters hippocampal function via the dysfunction of mitochondrial oxidative phosphorylation.     

Activation of the Interleukin-33/ST2 Pathway Exerts Deleterious Effects in Myxomatous Mitral Valve Disease

Mitral valve disease (MVD) is a frequent cause of heart failure and death worldwide, but its etiopathogenesis is not fully understood. Interleukin (IL)-33 regulates inflammation and thrombosis in the vascular endothelium and may play a role in the atherosclerotic process, but its role in mitral valve has not been investigated. We aim to explore IL-33 as a possible inductor of myxomatous degeneration in human mitral valves. We enrolled 103 patients suffering from severe mitral regurgitation due to myxomatous degeneration undergoing mitral valve replacement. Immunohistochemistry of the resected leaflets showed IL-33 and ST2 expression in both valve interstitial cells (VICs) and valve endothelial cells (VECs). Positive correlations were found between the levels of IL-33 and molecules implicated in the development of myxomatous MVD, such as proteoglycans, extracellular matrix remodeling enzymes (matrix metalloproteinases and their tissue inhibitors), inflammatory and fibrotic markers. Stimulation of single cell cultures of VICs and VECs with recombinant human IL-33 induced the expression of activated VIC markers, endothelial–mesenchymal transition of VECs, proteoglycan synthesis, inflammatory molecules and extracellular matrix turnover. Our findings suggest that the IL-33/ST2 system may be involved in the development of myxomatous MVD by enhancing extracellular matrix remodeling.     

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.     

Long-Term Severe In Vitro Hypoxia Exposure Enhances the Vascularization Potential of Human Adipose Tissue-Derived Stromal Vascular Fraction Cell Engineered Tissues

The therapeutic potential of mesenchymal stromal/stem cells (MSC) for treating cardiac ischemia strongly depends on their paracrine-mediated effects and their engraftment capacity in a hostile environment such as the infarcted myocardium. Adipose tissue-derived stromal vascular fraction (SVF) cells are a mixed population composed mainly of MSC and vascular cells, well known for their high angiogenic potential. A previous study showed that the angiogenic potential of SVF cells was further increased following their in vitro organization in an engineered tissue (patch) after perfusion-based bioreactor culture. This study aimed to investigate the possible changes in the cellular SVF composition, in vivo angiogenic potential, as well as engraftment capability upon in vitro culture in harsh hypoxia conditions. This mimics the possible delayed vascularization of the patch upon implantation in a low perfused myocardium. To this purpose, human SVF cells were seeded on a collagen sponge, cultured for 5 days in a perfusion-based bioreactor under normoxia or hypoxia (21% and <1% of oxygen tension, respectively) and subcutaneously implanted in nude rats for 3 and 28 days. Compared to ambient condition culture, hypoxic tension did not alter the SVF composition in vitro, showing similar numbers of MSC as well as endothelial and mural cells. Nevertheless, in vitro hypoxic culture significantly increased the release of vascular endothelial growth factor (p < 0.001) and the number of proliferating cells (p < 0.00001). Moreover, compared to ambient oxygen culture, exposure to hypoxia significantly enhanced the vessel length density in the engineered tissues following 28 days of implantation. The number of human cells and human proliferating cells in hypoxia-cultured constructs was also significantly increased after 3 and 28 days in vivo, compared to normoxia. These findings show that a possible in vivo delay in oxygen supply might not impair the vascularization potential of SVF- patches, which qualifies them for evaluation in a myocardial ischemia model.     

AMPK–mTOR Signaling and Cellular Adaptations in Hypoxia

Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I–IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed.     

缺氧诱导因子-1α与Wnt/β-catenin信号通路在缺氧胃癌细胞中的相互作用

目的在缺氧环境下,探讨缺氧诱导因子-1α(Hypoxia-inducible factor-1α,HIF-1α)与Wnt/β-catenin信号通路在胃癌细胞SGC-7901中的相互作用。方法将质粒β-catenin-micRNA转染SGC-7091细胞,筛选稳定转染细胞株SGC-7901-β-catenin-micRNA。将SGC-7901细胞分为对照组、脂质体组和阴性对照组,对照组:常氧培养48 h;脂质体组:常氧培养24 h后,加入10μl脂质体,培养24 h;阴性对照组:常氧培养24 h后,转染阴性对照质粒,培养24 h;设稳定转染细胞株为干扰组,常氧培养48 h。采用倍增时间试验、平板克隆形成试验及流式细胞术检测各组细胞倍增时间、平板克隆集落数及细胞周期的变化。另取SGC-7901细胞,分为对照组、缺氧组、双重缺氧组;对照组:常氧培养48 h;缺氧组:常氧培养32 h,物理缺氧培养16 h;双重缺氧组:常氧培养24 h,化学缺氧培养8 h后,再同时物理缺氧培养16 h。同时取稳定转染细胞株,分为对照干扰组、缺氧干扰组和双重缺氧干扰组。对照干扰组:常氧培养48 h;缺氧干扰组:常氧培养32 h,物理缺氧培养16 h;双重缺氧干扰组:常氧培养24 h,化学缺氧培养8 h后,再同时物理缺氧培养16 h。采用RT-PCR和Western blot法检测各组细胞中HIF-1α、β-catenin的mRNA及蛋白的表达水平。结果与对照组、脂质体组、阴性对照组比较,干扰组倍增时间延长,平板克隆集落数减少,细胞阻滞在G1期比例增加,S期减少,差异均有统计学意义(P<0.05)。缺氧组与对照组、双重缺氧组与缺氧组、缺氧干扰组与对照干扰组及双重缺氧干扰组与缺氧干扰组相比,HIF-1α、β-catenin的mRNA及蛋白表达水平均升高,差异有统计学意义(P均<0.05)。缺氧干扰组与缺氧组及双重缺氧干扰组与双重缺氧组比较,β-catenin、HIF-1α的mRNA及蛋白表达水平均降低,差异有统计学意义(P均<0.05)。结论 HIF-1α激活可调控Wnt/β-catenin通路,但也受控于Wnt/β-catenin通路,二者之间相互影响,相互调节。     

Radical Roles for RAGE in the Pathogenesis of Oxidative Stress in Cardiovascular Diseases and Beyond

Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.     

Hypoxic Conditions Promote the Angiogenic Potential of Human Induced Pluripotent Stem Cell-Derived Extracellular Vesicles

Stem cells secrete paracrine factors including extracellular vesicles (EVs) which can mediate cellular communication and support the regeneration of injured tissues. Reduced oxygen (hypoxia) as a key regulator in development and regeneration may influence cellular communication via EVs. We asked whether hypoxic conditioning during human induced pluripotent stem cell (iPSC) culture effects their EV quantity, quality or EV-based angiogenic potential. We produced iPSC-EVs from large-scale culture-conditioned media at 1%, 5% and 18% air oxygen using tangential flow filtration (TFF), with or without subsequent concentration by ultracentrifugation (TUCF). EVs were quantified by tunable resistive pulse sensing (TRPS), characterized according to MISEV2018 guidelines, and analyzed for angiogenic potential. We observed superior EV recovery by TFF compared to TUCF. We confirmed hypoxia efficacy by HIF-1α stabilization and pimonidazole hypoxyprobe. EV quantity did not differ significantly at different oxygen conditions. Significantly elevated angiogenic potential was observed for iPSC-EVs derived from 1% oxygen culture by TFF or TUCF as compared to EVs obtained at higher oxygen or the corresponding EV-depleted soluble factor fractions. Data thus demonstrate that cell-culture oxygen conditions and mode of EV preparation affect iPSC-EV function. We conclude that selecting appropriate protocols will further improve production of particularly potent iPSC-EV-based therapeutics.     

Lutein Protects RGC-5 Cells Against Hypoxia and Oxidative Stress

Retinal ischemia and oxidative stress lead to neuronal death in many ocular pathologies. Recently, we found that lutein, an oxy-carotenoid, protected the inner retina from ischemia/reperfusion injury. However, it is uncertain whether lutein directly protects retinal ganglion cells (RGCs). Here, an in vitro model of hypoxia and oxidative stress was used to further investigate the neuroprotective role of lutein in RGCs. Cobalt chloride (CoCl₂) and hydrogen peroxide (H₂O₂) were added to a transformed RGC cell line, RGC-5, to induce chemical hypoxia and oxidative stress, respectively. Either lutein or vehicle was added to cultured cells. A higher cell count was observed in the lutein-treated cells compared with the vehicle-treated cells. Our data from this in vitro model revealed that lutein might protect RGC-5 cells from damage when exposed to either CoCl₂-induced chemical hypoxia or H₂O₂-induced oxidative stress. These results suggest that lutein may play a role as a neuroprotectant.     

Nox2 Upregulation and p38α MAPK Activation in Right Ventricular Hypertrophy of Rats Exposed to Long-Term Chronic Intermittent Hypobaric Hypoxia

One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H₂O₂ concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.     

Obstructive Sleep Apnea as an Acceleration Trigger of Cellular Senescence Processes through Telomere Shortening

Obstructive sleep apnea (OSA) is chronic disorder which is characterized by recurrent pauses of breathing during sleep which leads to hypoxia and its two main pathological sequelae: oxidative stress and chronic inflammation. Both are also associated with cellular senescence. As OSA patients present with higher prevalence of age-related disorders, such as atrial hypertension or diabetes mellitus type 2, a relationship between OSA and accelerated aging is observable. Furthermore, it has been established that these OSA are associated with telomere shortening. This process in OSA is likely caused by increased oxidative DNA damage due to increased reactive oxygen species levels, DNA repair disruptions, hypoxia, chronic inflammation, and circadian clock disturbances. The aim of the review is to summarize study outcomes on changes in leukocyte telomere length (LTL) in OSA patients and describe possible molecular mechanisms which connect cellular senescence and the pathophysiology of OSA. The majority of OSA patients are characterized by LTL attrition due to oxidative stress, hypoxia and inflammation, which make a kind of positive feedback loop, and circadian clock disturbance.     

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 10⁸/mL versus 2 × 10⁷/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.     

Metabolomics of oxidative stress in recent studies of endogenous and exogenously administered intermediate metabolites

Aerobic metabolism occurs in a background of oxygen radicals and reactive oxygen species (ROS) that originate from the incomplete reduction of molecular oxygen in electron transfer reactions. The essential role of aerobic metabolism, the generation and consumption of ATP and other high energy phosphates, sustains a balance of approximately 3000 essential human metabolites that serve not only as nutrients, but also as antioxidants, neurotransmitters, osmolytes, and participants in ligand-based and other cellular signaling. In hypoxia, ischemia, and oxidative stress, where pathological circumstances cause oxygen radicals to form at a rate greater than is possible for their consumption, changes in the composition of metabolite ensembles, or metabolomes, can be associated with physiological changes. Metabolomics and metabonomics are a scientific disciplines that focuse on quantifying dynamic metabolome responses, using multivariate analytical approaches derived from methods within genomics, a discipline that consolidated innovative analysis techniques for situations where the number of biomarkers (metabolites in our case) greatly exceeds the number of subjects. This review focuses on the behavior of cytosolic, mitochondrial, and redox metabolites in ameliorating or exacerbating oxidative stress. After reviewing work regarding a small number of metabolites-pyruvate, ethyl pyruvate, and fructose-1,6-bisphosphate-whose exogenous administration was found to ameliorate oxidative stress, a subsequent section reviews basic multivariate statistical methods common in metabolomics research, and their application in human and preclinical studies emphasizing oxidative stress. Particular attention is paid to new NMR spectroscopy methods in metabolomics and metabonomics. Because complex relationships connect oxidative stress to so many physiological processes, studies from different disciplines were reviewed. All, however, shared the common goal of ultimately developing "omics"-based, diagnostic tests to help influence therapies.