Differential protein expression in brain capillary endothelial cells induced by hypoxia and posthypoxic reoxygenation

Cerebral ischemia causes functional alteration of the blood-brain barrier, formed by brain capillary endothelial cells (BCEC). Changes in protein expression and activity of selected differentially expressed enzymes were investigated in BCEC subjected to hypoxia (24 h) alone or followed by a 24-h reoxygenation. BCEC proteins were isolated, separated by 2-DE, and identified by MALDI-MS. Computer-based 2-D gel analysis identified 21 up-regulated proteins and 4 down-regulated proteins after hypoxia alone and 9 proteins that were further up-regulated after posthypoxic reoxygenation. The expression of the majority of hypoxia-induced proteins was reduced toward control levels during reoxygenation. The most prominent changes were identified for glycolytic enzymes (e.g., phosphoglycerate kinase), proteins of the ER (e.g., calreticulin), and cytoskeletal (e.g., vimentin) proteins. The results indicate that BCEC respond to hypoxia/reoxygenation by adaptive up-regulation of proteins involved in the glycolysis, protein synthesis, and stress response.     

Tempol modulates changes in xenobiotic permeability and occludin oligomeric assemblies at the blood-brain barrier during inflammatory pain

Our laboratory has shown that λ-carrageenan-induced peripheral inflammatory pain (CIP) can alter tight junction (TJ) protein expression and/or assembly leading to changes in blood-brain barrier xenobiotic permeability. However, the role of reactive oxygen species (ROS) and subsequent oxidative stress during CIP is unknown. ROS (i.e., superoxide) are known to cause cellular damage in response to pain/inflammation. Therefore, we examined oxidative stress-associated effects at the blood-brain barrier (BBB) in CIP rats. During CIP, increased staining of nitrosylated proteins was detected in hind paw tissue and enhanced presence of protein adducts containing 3-nitrotyrosine occurred at two molecular weights (i.e., 85 and 44 kDa) in brain microvessels. Tempol, a pharmacological ROS scavenger, attenuated formation of 3-nitrotyrosine-containing proteins in both the hind paw and in brain microvessels when administered 10 min before footpad injection of λ-carrageenan. Similarly, CIP increased 4-hydroxynoneal staining in brain microvessels and this effect was reduced by tempol. Brain permeability to [(14)C]sucrose and [(3)H]codeine was increased, and oligomeric assemblies of occludin, a critical TJ protein, were altered after 3 h CIP. Tempol attenuated both [(14)C]sucrose and [(3)H]codeine brain uptake as well as protected occludin oligomers from disruption in CIP animals, suggesting that ROS production/oxidative stress is involved in modulating BBB functional integrity during pain/inflammation. Interestingly, tempol administration reduced codeine analgesia in CIP animals, indicating that oxidative stress during pain/inflammation may affect opioid delivery to the brain and subsequent efficacy. Taken together, our data show for the first time that ROS pharmacological scavenging is a viable approach for maintaining BBB integrity and controlling central nervous system drug delivery during acute inflammatory pain.     

Transendothelial permeability changes induced by free radicals in an in vitro model of the blood-brain barrier.

In the present study, we investigated the changes in blood-brain barrier (BBB) permeability following brain endothelial cell exposure to different xenobiotics able to promote free radical generation during their metabolism. Our in vitro BBB model consisted of confluent monolayers of immortalized rat brain capillary endothelial cells (RBE4) grown on collagen-coated filters in the presence of C6 glioma cells grown in the lower compartment. We have recently shown that a range of xenobiotics, including menadione, nitrofurazone, and methylviologen (paraquat) may undergo monoelectronic redox cycling in isolated brain capillaries, giving rise to reactive oxygen species. In this study, addition of 100 microM menadione to the culture medium for 30 min significantly increased the permeability of endothelial cell monolayers to radiolabeled sucrose. The effect on endothelial permeability induced by menadione was dose-dependent and reversible. These permeability changes preceded the onset of cell death, as assessed by the Trypan blue exclusion method. Pre-incubation with superoxide dismutase and catalase blocked changes in sucrose permeability to control levels in a dose-dependent manner, suggesting the involvement of reactive oxygen species in menadione-induced BBB opening.     

Zinc modulates cytokine-induced lung epithelial cell barrier permeability

Apoptosis plays a causative role in acute lung injury in part due to epithelial cell loss. We recently reported that zinc protects the lung epithelium during inflammatory stress whereas depletion of intracellular zinc enhances extrinsic apoptosis. In this investigation, we evaluated the relationship between zinc, caspase-3, and cell-to-cell contact via proteins that form the adherens junction complex. Cell adhesion proteins are directly responsible for formation of the mechanical barrier of the lung epithelium. We hypothesized that exposure to inflammatory cytokines, in conjunction with zinc deprivation, would induce caspase-3, leading to degradation of junction proteins, loss of cell-to-cell contact, and compromised barrier function. Primary human upper airway and type I/II alveolar epithelial cultures were obtained from multiple donors and exposed to inflammatory stimuli that provoke extrinsic apoptosis in addition to depletion of intracellular zinc. We observed that zinc deprivation combined with tumor necrosis factor-alpha, interferon-gamma, and Fas receptor ligation accelerates caspase-3 activation, proteolysis of E-cadherin and beta-catenin, and cellular apoptosis, leading to increased paracellular leak across monolayers of both upper airway and alveolar lung epithelial cultures. Zinc supplementation inhibited apoptosis and paracellular leak, whereas caspase inhibition was less effective. We conclude that zinc is a vital factor in the lung epithelium that protects against death receptor-mediated apoptosis and barrier dysfunction. Furthermore, our findings suggest that although caspase-3 inhibition reduces lung epithelial apoptosis it does not prevent mechanical dysfunction. These findings facilitate future studies aimed at developing therapeutic strategies to prevent acute lung injury.     

Homocysteine in microvascular endothelial cell barrier permeability

Redox stress activates the endothelium and upregulates matrix metalloproteinases (MMPs), which degrade the matrix and lead to blood-endothelial barrier leakage. Interestingly, elevated levels of plasma homocysteine (Hcy) are associated with vascular dementia, seizure, stroke, and Alzheimer disease. Hcy competes with the γ-aminobutyric acid (GABA)-A/B receptors and behave like an excitatory neurotransmitter. GABA stimulates the inhibitory neurotransmitter GABA-A/B receptor and decreases arterial blood pressure. However, the neural mechanisms of microvascular remodeling in hyperhomocysteinemia are unclear. This review addresses the idea that Hcy induces microvascular permeability by attenuating the GABA-A/B receptors and increasing redox stress, which activates a disintegrin and metalloproteinase that suppresses tissue inhibitors of metalloproteinase. This process causes disruption of the matrix in the blood-brain barrier. Understanding the mechanism of Hcy-mediated changes in permeability of the blood-brain barrier and extracellular matrix that can alter the neuronal environment in cerebral-vascular dementia is of great importance in developing treatments for this disease.     

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O₂, 5% CO₂, 93% N₂) for 24 h followed by reoxygenation (21% O₂, 5% CO₂, 74% N₂) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by ～30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.     

Erythropoietin prevents the increase in blood-brain barrier permeability during pentylentetrazol induced seizures

Recombinant human erythropoietin (r-Hu EPO) has been shown to exert neuroprotection in ischemic, excitotoxicity, trauma, convulsions and neurodegenerative disorders. Blood-brain barrier (BBB) leakage plays a role in the pathogenesis of many pathological states of the brain including neurodegenerative disorders. This study aimed to investigate the effects of r-Hu EPO on BBB integrity in pentylentetrazol (PTZ) induced seizures in rats. Seizures were observed and evaluated regard to latency and intensity for an hour. Macroscopical and spectrophotometrical measurement of Evans Blue (EB) leakage were observed for BBB integrity. r-Hu EPO was given intraperitoneally 24 h prior to seizure induction. Total seizure duration of 720+/-50 s after single PTZ administration (80 mg/kg i.p.) was declined to 190+/-40 s in r-Hu EPO pretreatment. A typical BBB breakdown pattern (i.e. staining in cerebellum, cerebral cortex, midbrain, hippocampus, thalamus and corpus striatum) was observed in rat brains with PTZ induced seizures; whereas, EPO pretreatment confined BBB leakage to cerebellum and cortical areas, and lessened the intensity of tonic-clonic seizures observed in PTZ seizures. The protective effect of r-Hu EPO on BBB permeability in seizures is a new and original finding. The protective action of r-Hu EPO in seizures and some of CNS pathologies warrant further investigations.     

Cathelicidin LL-37 peptide regulates endothelial cell stiffness and endothelial barrier permeability

LL-37 peptide is a multifunctional host defense molecule essential for normal immune responses to infection or tissue injury. In this study we assess the impact of LL-37 on endothelial stiffness and barrier permeability. Fluorescence microscopy reveals membrane localization of LL-37 after its incubation with human umbilical vein endothelial cells (HUVECs). A concentration-dependent increase in stiffness was observed in HUVECs, bovine aortic endothelial cells (BAECs), human pulmonary microvascular endothelial cells, and mouse aorta upon LL-37 (0.5-5 μM) addition. Stiffening of BAECs by LL-37 was blocked by P2X7 receptor antagonists and by the intracellular Ca2(+) chelator BAPTA-AM. Increased cellular stiffness correlated with a decrease in permeability of HUVEC cell monolayers after LL-37 addition compared with nontreated cells, which was similar to the effect observed upon treatment with sphingosine 1-phosphate, and both treatments increased F-actin content in the cortical region of the cells. These results suggest that the antiinflammatory effect of LL-37 at the site of infection or injury involves an LL-37-mediated increase in cell stiffening that prevents increased pericellular permeability. Such a mechanism may help to maintain tissue fluid homeostasis.     

Inhibition of angiotensin-converting enzyme protects endothelial cell against hypoxia/reoxygenation injury

Cardiovascular tissue injury in ischemia/reperfusion has been shown to be prevented by angiotensin-converting enzyme (ACE) inhibitors. However, the mechanism on endothelial cells has not been assessed in detail. Cultured human aortic endothelial cells (HAEC) were exposed to hypoxia with or without reoxygenation. Hypoxia enhanced apoptosis along with the activation of caspase-3. Reoxygenation increased lactate dehydrogenase release time-dependently, along with an increase of intracellular oxygen radicals. ACE inhibitor quinaprilat and bradykinin significantly lessened apoptosis and lactate dehydrogenase release with these effects being diminished by a kinin B2 receptor antagonist and a nitric oxide synthase inhibitor. In conclusion, hypoxia activated the suicide pathway leading to apoptosis of HAEC by enhancing caspase-3 activity, while subsequent reoxygenation induced necrosis by enhancing oxygen radical production. Quinaprilat could ameliorate both apoptosis and necrosis through the upregulation of constitutive endothelial nitric oxide synthase via an increase of bradykinin, with the resulting increase of nitric oxide.     

Phase changes in the permeability of the mouse blood-brain barrier after uneven gamma-irradiation

Two phases of changes in blood-brain-barrier permeability for sour fuchsin have been observed in the early period after head or body irradiation at 2.58 C/kg: an increase (up to 2 h) and a decrease (from 2 to 6 h). The correlation between the decrease in BBB permeability and the frequency of clinical manifestations of radiation disease has been established. BBB permeability progressively increased later after head irradiation (24-120 h) which was indicative of its absolute dysfunction. The changes observed may reflect redistribution of fluid and electrolytes in the vascular bed and tissues leading to the onset of brain tissue edema.     

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549

Active oxygen species are generated in cells during pathophysiologic conditions such as illflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We inves-tigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K⁺ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K⁺) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K⁺ channels are activated following reoxygenation but not during the hypoxia phase. The K⁺ currents decayed with time following reoxygenation. The decay characteristics of the K⁺ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K⁺ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K⁺ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K⁺ currents. © 1993 Wiley-Liss, Inc.     

MsrA protects cardiac myocytes against hypoxia/reoxygenation induced cell death

Reactive oxygen species (ROS) are critical in tissue responses to ischemia-reperfusion. The enzyme methionine sulfoxide reductase-A (MsrA) is capable of protecting cells against oxidative damage by reversing damage to proteins caused by methionine oxidation or by decreasing ROS through a scavenger mechanism. The current study employed adenovirus mediated over-expression of MsrA in primary neonatal rat cardiac myocytes to determine the effect of this enzyme in protecting against hypoxia/reoxygenation in this tissue. Cells were transduced with MsrA encoding adenovirus and subjected to hypoxia/reoxygenation. Apoptotic cell death was decreased by greater than 45% in cells over-expressing MsrA relative to cells transduced with a control virus. Likewise total cell death as determined by levels of LDH release was dramatically decreased by MsrA over-expression. These observations indicate that MsrA is protective against hypoxia/reoxygenation stress in cardiac myocytes and point to MsrA as an important therapeutic target for ischemic heart disease.     

Gap junctional permeability is affected by cell volume changes and modulates volume regulation

Isolated pancreatic acinar cell pairs became electrically uncoupled by exposure to a mild hypotonic shock. Reduction of bath osmolarity caused a delayed closure of gap junctional channels in the minute range. Dialysis of cell pairs by GTP[S] in the double whole-cell patch-clamp mode shortened the latency and shifted the hypotonically induced electrical uncoupling to lower osmolarity changes. Cellular treatment with cytochalasin B catalyzed electrical uncoupling by a hypotonic shock. In all cases, electrical uncoupling could be blocked completely by the protein kinase C (PKC) inhibitor polymyxin B. These results provide the first evidence suggesting that changes of cell volume and gap junctional permeability are correlated and that a G-protein dependent mechanism is involved. Evidence is presented that gap junctional coupling modulates volume regulation.     

Neutrophil tolerance to oxidative stress induced by hypoxia/reoxygenation

Repetitive episodes of hypoxia/reoxygenation induce cellular adaptations resulting in a tolerance process against oxidative stress. We studied the effects of chronic episodes of hypoxia/reoxygenation on neutrophil antioxidant defenses, neutrophil oxidative capability, and oxidative damage induced in neutrophils and plasma. Seven professional apnea divers participated in the study. Blood samples were taken under basal conditions, after a diving apnea session, and under basal conditions after five consecutive days of diving apnea sessions (basal post-diving). Chronic episodes of hypoxia/reoxygenation increased malondialdehyde (MDA), carbonyl derivates and creatine kinase (CPK) in plasma. Neutrophil catalase (CAT) levels were higher in basal post-diving. Neutrophil oxidative burst was maintained after diving, although the maximum response was delayed in basal post-diving. Neutrophil thioredoxin reductase (TR) activity increased in basal post-diving, and glutathione reductase (GR) activity was maintained. Chronic, repetitive episodes of diving apnea induce neutrophil adaptations in order to delay the oxidative burst response and to facilitate protein reduction. Diving apnea could be a good model to study tolerance to the oxidative stress generated by hypoxia/reoxygenation.     

Rac1蛋白活化加速缺氧诱导的人血管内皮细胞衰老

为阐明Rac1蛋白在人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）衰老中的作用及分子机制,我们采用持续缺氧的方法诱导内皮细胞衰老,检测缺氧前后内皮细胞衰老标志基因SA-β-Gal和PAI-1的表达、细胞周期分布和细胞增殖情况,同时分析缺氧前后细胞内Rac1蛋白的表达.结果显示,持续缺氧96 h后,HUVECs体积变大,细胞浆内颗粒和空泡增多,SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞发生G1期阻滞,细胞增殖受抑,活化型Rac1蛋白表达上调,提示持续缺氧诱导的内皮细胞衰老可能与Rac1蛋白的活化有关.为进一步明确内皮细胞衰老与Rac1蛋白的关系,应用逆转录病毒将持续活化型Rac1（V12Rac1）和主导抑制型Rac1（N17Rac1）基因分别瞬时感染HUVECs,比较三种HUVECs（HUVECs,V12Rac1-HUVECs,N17Rac1-HUVECs）缺氧后的衰老变化,并分析其下游调控分子--血清反应因子（serum response factor,SRF）的表达和定位变化.研究发现,缺氧培养V12Rac1-HUVECs 48 h即可引起细胞衰老,表现为SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞出现明显的G1期阻滞并且细胞增殖受抑,其改变与缺氧96 h的HUVECs相似;而N17Rac1明显抑制缺氧引起的内皮细胞衰老发生.上述结果说明,Rac1蛋白活化可以加速缺氧诱导的内皮细胞衰老,而抑制Rac1蛋白的活性则可抑制缺氧诱导的内皮细胞衰老.为进一步研究Rac1蛋白引起内皮细胞衰老的机制,通过免疫荧光染色及Western blot分析检测三种细胞缺氧处理后SRF的表达,发现：与HUVECs细胞比较,V12Rac1引起缺氧48 h HUVECs核蛋白中SRF的表达明显下降,SRF入核转位受到明显抑制;而N17Rac1感染后,缺氧HUVECs细胞核蛋白中SRF表达明显增多.上述结果提示：缺氧状态下Rac1蛋白活化能够明显加速HUVECs衰老,而抑制Rac1蛋白活性则明显抑制缺氧诱导的HUVECs衰老,SRF蛋白的核转位活化参与了Rac1蛋白调控HUVECs衰老的发生.     

PKC and RhoA signals cross-talk in Escherichia coli endotoxin induced alterations in brain endothelial permeability

Escherichia coli endotoxin LPS regulates blood-brain barrier permeability by disrupting the tight junction (TJ) complex between brain endothelial cells. This study used Bend.3 cells to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. The LPS-induced alterations in the brain endothelial barrier were associated with PKC (a, β, ζ) and RhoA, but were independent of PI3K and the tyrosine kinase pathway. Inhibition of PKC (a, β, ζ) and RhoA activity using shRNA and dominant negative mutants diminished the effects of LPS on the brain's endothelial TJs. The interactions between the PKC and Rho pathways were therefore examined. PKC-a and PKC-ζ, but not PKC-β interacted with RhoA in Bend.3 cells stimulated by LPS. PKC-a acted as the upstream molecule for Rho and PKC-ζ acted as the downstream target for Rho. Comparing the effect of double inhibition of "Rho and PKC" and single inhibition of "Rho" or "PKC" confirmed that this interaction is critical for LPS-induced brain endothelial cell hyperpermeability. Collectively these data are the first to suggest that LPS affects the brain's endothelial TJ barrier via PKC (a, β, ζ)- and RhoA, independent of the PI3K and tyrosine kinase pathways. In addition, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulator for RhoA in the process.