Reoxygenation of endothelial cells increases permeability by oxidant-dependent mechanisms (original) (raw)
Related papers
Free Radical Biology and Medicine, 1990
Ahall'act-We assessed the effect of hypoxia/reoxygenation on ~4C-albumin flux across endothelial monolayers. Cultured bovine pulmonary artery endothelial cells were grown to confluence on nitrocellulose filters (pore size 12 p,m). The endothelialized filters were mounted in Ussing-type chambers which were filled with cell culture medium (M 199). Equimolar amounts (33 nM) of 14C-labeled and unlabeled albumin were added to the "hot" and "cold" chambers, respectively. The monolayers were then exposed to successive periods (90 min) of normoxia (pO 2 145 mmHg), hypoxia (pO 2 20 mmHg), and reoxygenation (pO 2 145 mmHg). A gas bubbling system was used to control media pO 2 and to ensure adequate mixing. Four aliquots of culture media were taken during each period in order to calculate the t4C-albumin permeability across the endothetialized filter. In some experiments, either the xanthine oxidase inhibitor, oxypurinol (10 o.M), or superoxide dismutase (600 U/mL), was added to the media immediately prior to the experiments. As compared to the normoxic control period, albumin permeability was 1.5 times higher during hypoxia (p<0.01) and 2.3 times higher during reoxygenation (p<0.01). The reoxygenation-induced increase in albumin permeability was prevented by either oxypurinol or superoxide dismutase. These data indicate that xanthine oxidase-derived oxygen radicals contribute to the hypoxia/reoxygenation-induced endothelial cell dysfunction. The altered endothelial barrier function induced by hypoxia/reoxygenation is consistent with the microvaseular dysfunction observed following repeffusion of ischemic tissues.
American Journal of Respiratory Cell and Molecular Biology, 2003
Hypoxic preconditioning is protective against oxidant-related thelial survival and growth are likely to be involved in the damage in various organs, such as the heart. We previously pathogenesis of BPD. Hyperoxia can induce injury and showed that rats exposed to hypoxia also exhibit resistance to death of pulmonary capillary endothelial, as well as epithelethal pulmonary oxygen toxicity. The underlying mechanism lial cells (4, 5). and whether similar preconditioning is applicable to cellular Endothelial cells thrive in hypoxic environments in which models is unknown. In the present study, it was found that they can better maintain their integrity, survive, replicate, hypoxic pre-exposure induces a significant protective effect and form capillary networks (6, 7). Paradoxically, exposures against hyperoxia-induced cell death in human lung microvasto hypoxia can precondition various organs, including the cular endothelial cells (HLMVECs) and epithelial type II-like heart, brain, kidney, liver, and skeletal muscle, protecting A549 cells. This effect of hypoxia is mediated by the phosphatidylinositol 3-kinase (PI3-K) signaling pathway because the pres-them against subsequent oxidative stress such as that caused ence of the PI3-K inhibitors, LY294002 and wortmannin, during by ischemia-reperfusion. Therefore, it is reasonable to anticpre-exposure to hypoxia completely blocks subsequent protecipate that such preconditioning in cultured lung cells might tion. Further, the hypoxia-dependent protection from hyperbe protective against hyperoxic injury. Previously, our studoxia was found to be associated with a 2-fold increase in PI3-K ies with rats acclimated to hypoxia have shown consistent activity in hypoxia. Transient overexpression of a catalytically (100%) and prolonged survival during subsequent hyperactive class IA PI3-K p110␣ isoform also enhanced survival of oxic exposure which is almost uniformly fatal to 21% oxy-A549 cells 2-fold compared with the empty vector control. gen-pre-exposed control animals (8). Protection against ox-These results indicate that hypoxia-induced activation of PI3-K idative stress by hypoxic preconditioning has not been is an important event in the acquisition of resistance against subsequent hyperoxic toxicity.
Role of Mitochondrial Oxidant Generation in Endothelial Cell Responses to Hypoxia
Arteriosclerosis, Thrombosis, and Vascular Biology, 2002
Endothelial cells increase their secretion of the cytokine interleukin-6 (IL-6) during hypoxia, which then acts in an autocrine fashion to increase the permeability of cell monolayers. These responses are attenuated by antioxidants, suggesting that reactive oxygen species (ROS) participate in signaling in hypoxic endothelium. We tested whether mitochondria are responsible for these ROS in human umbilical vein endothelial cells exposed to hypoxia. Oxidation of the probe 2Ј,7Ј-dichlorodihydrofluorescein to fluorescent dichlorofluorescein or the probe dihydroethidium was used to assess oxidant signaling, whereas permeability was assessed by using transendothelial electrical resistance. Hypoxia elicited increases in dichlorofluorescein and dihydroethidium fluorescence that were abrogated by the mitochondrial electron transport (ET) inhibitors rotenone (2 mol/L) and diphenyleneiodonium (5 mol/L). The same ET inhibitors also attenuated hypoxia-induced increases in nuclear factor-B (NF-B) activation, although they did not abrogate NF-B activation in response to endotoxin (lipopolysaccharide). ET inhibition also abolished the hypoxia-induced increases in IL-6 mRNA expression, hypoxia-stimulated IL-6 secretion into the media, and the hypoxia-induced increases in transendothelial electrical resistance of human umbilical vein endothelial cell monolayers. By contrast, the above responses to hypoxia were not significantly affected by treatment with the NAD(P)H oxidase inhibitor apocynin (30 mol/L), the xanthine oxidase inhibitor allopurinol (100 mol/L), or the NO synthase inhibitor N-nitro-L-arginine (100 mol/L). We conclude that ROS signals originating from the mitochondrial ET chain trigger the increase in NF-B activation, the transcriptional activation of IL-6, the secretion of IL-6 into the cell culture media, and the increases in endothelial permeability observed during hypoxia. (Arterioscler Thromb Vasc Biol. 2002;22:566-573.)
Journal of Cellular Physiology, 1991
In bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose-dependent manner at lower oxygen concentrations, as progression into S phase from GI was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a -3-fold increase in the total number of high-affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia-mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the rnonolayers consisted of larger cells with altered assembly of the actin-based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monola ers to macromolecules and lower molecular weight solutes anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that microand macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endotheliurn exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions. was increased. Ce Y I surface coagulant properties were also perturbed: the Hypoxia often occurs during regeneration of endothelium, such as takes place after vascular injury, or during angiogenesis in wound repair or neovascularization of tumors. In each of these situations, endothelial cell (EC) growth, and formation of a monolayer regulating permeability and coa lation, must occur for blood flow to be initiated an Y maintained. These considerations led us to examine the effect of h poxia thelium.
Cellular Physiology and Biochemistry, 2010
Under hypoxic conditions eukaryotic cells and tissues undergo adaptive responses involving glycolysis, angiogenesis, vasoconstriction and inflammation. The underlying molecular mechanisms are not yet fully elucidated and are most likely cell and tissue specific. In the lung, alveolar epithelial cells and microvascular endothelial cells are highly sensitive to hypoxia and together orchestrate a rapid and sustained adaptive response. We examined the effect of different oxygen tensions on cell viability, glucose metabolism, key transcription factors and signaling molecules, in alveolar epithelial cells (A549) and microvascular endothelial cells (HMEC-1). Both cell types tolerated hypoxia without detectable cell injury. Hypoxia induced glycolysis in both epithelial and microvascular endothelial cells, although A549 cells exhibited a higher rate of glucose consumption. The transcription factor CREB (cAMP response element binding protein) was activated with decreasing oxygen tensions in both cell types. This effect was again more marked in A549 cells, demonstrating epithelial cells to be more oxygen sensitive. Activating Transcription Factor 3 (ATF-3) was heavily induced by hypoxia in A549 cells but not in HMEC-1 cells. Both cell types exhibited hypoxia induced secretion of VEGF and IL-6. Secretion of the vasoconstrictor endothelin-1 (ET1) was increased by hypoxia in HMEC-1 cells but decreased in A549 cells. These data reveal that both cell types exhibit an adaptive response to hypoxia but alveolar epithelial cells are generally more sensitive. ET-1 was oppositely regulated by decreased oxygen tensions in the investigated cell types. The present study further elucidates the adaptive molecular mechanisms in pulmonary hypoxia and demonstrates cell specific responses.
Biochemical and morphological changes in endothelial cells in response to hypoxic interstitial edema
Respiratory Research, 2006
Background: A correlation between interstial pulmonary matrix disorganization and lung cellular response was recently documented in cardiogenic interstitial edema as changes in the signal-cellular transduction platforms (lipid microdomains: caveoale and lipid rafts). These findings led to hypothesize a specific "sensing" function by lung cells resulting from a perturbation in cell-matrix interaction. We reason that the cell-matrix interaction may differ between the cardiogenic and the hypoxic type of lung edema due to the observed difference in the sequential degradation of matrix proteoglycans (PGs) family. In cardiogenic edema a major fragmentation of high molecular weight PGs of the interfibrillar matrix was found, while in hypoxia the fragmentation process mostly involved the PGs of the basement membrane controlling microvascular permeability. Based on these considerations, we aim to describe potential differences in the lung cellular response to the two types of edema.
Effects of hypoxia on the pulmonary microvascular permeability in adult rabbit lung
Journal of Biomedical Science, 1995
Within the last 30 years, researchers have explored what role hypoxia might play in causing permeability changes in the pulmonary microvasculature. Since the data accumulated thus tar are unclear, the effects of hypoxia on microvascular transport in the isolated, Ringer's perfused adult rabbit lung was observed and the following parameters were measured or computed for both oxygenated and hypoxic perfusates: pulmonary arterial (ra) and pulmonary venous (r0 resistances, pulmonary capillary filtration coefficients (Kf), and pulmonary capillary endothelial reflection coefficients ((;) for NaCI and inulin. Separate reservoir bottles were used to create the desired oxygenated (aeration of solution with 95% 02-5% CO2) gas mixture or hypoxic (aeration of solution with 95% N2-5% C02) gas mixture. A higher, but not significant, resistance value was found during the oxygenated state. A significant increase in the pulmonary capillary filtration coefficient during hypoxia (10.72 x 10-4 +-0.446 x 4 3 10-cm/s cm H20 for the h3qgoxic perfusate and 8.80 x 10-4 + 0.384 x 10-4 cm3/s cm H20 for the oxygenated perfusate) was found and a significant difference between oxygenated and hypoxic pulmonary capillary reflection coefficients for inulin was computed (oxygenated solution revealed a finding of 0.120 + 0.003 and the hypoxic solution revealed 0.105-+ 0.002). These findings imply a change in the microvaseular permeability during hypoxia. According to the pore theory, a change in pore number, pore size, or both could have occurred. However, from the reflection coefficient data, a change in pore radius seems most likely.
Antioxidants & Redox Signaling, 2004
XYGEN is an important modulator of cellular functions in both normal physiology and disease states. Cells respond to oxygen over a wide range of concentrations from anoxia to hyperoxia. Baseline metabolism and function typically occur in normoxic environments (30-90 mm Hg of O 2) and can modulate differentiated cell functions (15). Hyperoxic conditions often result in the generation of reactive oxygen species that have been implicated in cell injury via lipid peroxidation and cytokine expression (5). In lieu of such diversity in cellular responses to oxygen, the dynamics of tissue oxygenation, including the transport of oxygen and the possible existence of an oxygen gradient across the cell membrane, 597
Frontiers in Physiology, 2018
Intermittent hypoxia is a major factor in clinical conditions like the obstructive sleep apnea syndrome or the cyclic recruitment and derecruitment of atelectasis in acute respiratory distress syndrome and positive pressure mechanical ventilation. In vivo investigations of the direct impact of intermittent hypoxia are frequently hampered by multiple co-morbidities of patients. Therefore, cell culture experiments are important model systems to elucidate molecular mechanisms that are involved in the cellular response to alternating oxygen conditions and could represent future targets for tailored therapies. In this study, we focused on mouse lung endothelial cells as a first frontier to encounter altered oxygen due to disturbances in airway or lung function, that play an important role in the development of secondary diseases like vascular disease and pulmonary hypertension. We analyzed key markers for endothelial function including cell adhesion molecules, molecules involved in regulation of fibrinolysis, hemostasis, redox balance, and regulators of gene expression like miRNAs. Results show that short-time exposure to intermittent hypoxia has little impact on vitality and health of cells. At early timepoints and up to 24 h, many endothelial markers are unchanged in their expression and some indicators of injury are even downregulated. However, in the long-term, multiple signaling pathways are activated, that ultimately result in cellular inflammation, oxidative stress, and apoptosis.
Hypoxia attenuates effector-target cell interaction in the airway and pulmonary vascular compartment
Clinical & Experimental Immunology, 2007
Leucocyte infiltration is known to play an important role in hypoxia-induced tissue damage. However, little information is available about hypoxia and interaction of effector (neutrophils) with target cells (alveolar epithelial cells, AEC; rat pulmonary artery endothelial cells, RPAEC). The goal of this study was to elucidate hypoxia-induced changes of effector-target cell interaction. AEC and RPAEC were exposed to 5% oxygen for 2-6 h. Intercellular adhesion molecule-1 (ICAM-1) expression was determined and cell adherence as well as cytotoxicity assays were performed. Nitric oxide and heat shock protein 70 (HSP70) production was assessed in target cells. Under hypoxic conditions enhanced ICAM-1 production was found in both cell types. This resulted in an increase of adherent neutrophils to AEC and RPAEC. The death rate of hypoxia-exposed target cells decreased significantly in comparison to control cells. Nitric oxide (NO) concentration was enhanced, as was production of HSP70 in AEC. Blocking NO production in target cells resulted in increased cytotoxicity in AEC and RPAEC. This study shows for the first time that target cells are more resistant to effector cells under hypoxia, suggesting hypoxiainduced cell protection. An underlying mechanism for this phenomenon might be the protective effect of increased levels of NO in target cells.