HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-alpha in cerebral vascular endothelial cells

Tumor necrosis factor- (TNF-) causes oxidative stress and apoptosis in a variety of cell types. Heme oxygenase (HO) degrades heme to bilirubin, an antioxidant, and carbon monoxide (CO), a cell cycle modulator, and a vasodilator. Newborn pig cerebral microvascular endothelial cells (CMVEC) highly express constitutive HO-2. We investigated the role of HO-2 in protection against TNF--induced apoptosis in cerebral vascular endothelium. In CMVEC from mice and newborn pigs, 15 ng/ml TNF- alone, or with 10 µg/ml cycloheximide (CHX) caused apoptosis detected by nuclear translocation of p65 NF-B, caspase-3 activation, DNA fragmentation, cell-cell contact destabilization, and cell detachment. TNF- did not induce HO-1 expression in CMVEC. CMVEC from HO-2 knockout mice showed greater sensitivity to apoptosis caused by serum deprivation and TNF- than did wild-type mice. TNF- increased reactive oxygen species generation, including hydrogen peroxide and superoxide radicals, as detected by dihydrorhodamine-123 and dihydroethidium. The TNF- response was inhibited by superoxide dismutase and catalase suggesting apoptosis is oxidative stress related. Inhibition of endogenous HO-2 in newborn pig CMVEC increased oxidative stress and exaggerated apoptosis caused by serum deprivation and TNF-. In HO-1-overexpressing CMVEC (HO-1 selective induction by cobalt portophyrin), TNF- did not cause apoptosis. A CO-releasing compound, CORM-A1, and bilirubin blocked TNF--induced reactive oxygen species accumulation and apoptosis consistent with the antioxidant and antiapoptotic roles of the end products of HO activity. We conclude that HO-2 is critical for protection of cerebrovascular endothelium against apoptotic changes induced by oxidative stress and cytokine-mediated inflammation. carbon monoxide; bilirubin; vascular injury; reactive oxygen species; heme oxygenase; cycloheximide     

Papaverine induces apoptosis in vascular endothelial and smooth muscle cells

Papaverine is a vasodilator commonly used in the treatment of vasospasmic diseases such as cerebral spasm associated with subarachnoid hemorrhage, and in the prevention of spasm of coronary artery bypass graft by intraluminal and/or extraluminal administration. In this study, we examined whether papaverine in the range of concentrations used clinically causes apoptosis of vascular endothelial and smooth muscle cells. Apoptotic cells were identified by morphological changes and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. In porcine coronary endothelial cells (EC) and rat aortic smooth muscle cells (SMC), papaverine at the concentration of 10(-3) M induced membrane blebbing within 1 hour of incubation. Nuclear condensation and fragmentation were found after 24 hours of treatment. The number of apoptotic cells stained with the TUNEL method was significantly higher in the EC and the SMC after 24 hours of incubation with papaverine at the concentrations of 10(-4) and 10(-3) M than their respective controls. Acidified saline solution (pH 4.8, as control for 10(-3) M papaverine hydrochloride) did not cause apoptosis in these cells. These results showed that papaverine could damage endothelial and smooth muscle cells by inducing changes which are associated with events leading to apoptosis. Since integrity of endothelial cells is critical for normal vascular function, vascular administration of papaverine for clinical use, especially at high concentrations (> or = 10(-4) M), should be re-considered.     

Nitric oxide induces oxidative stress and apoptosis in neuronal cells

Within the central nervous system and under normal conditions, nitric oxide (NO) is an important physiological signaling molecule. When produced in large excess, NO also displays neurotoxicity. In our previous report, we have demonstrated that the exposure of neuronal cells to NO donors induced apoptotic cell death, while pretreatment with free radical scavengers L-ascorbic acid 2-[3, 4-dihydro-2,5,7,8-tetramethyl-2-(4,8, 12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K1) or superoxide dismutase attenuated apoptosis effectively, suggesting that reactive oxygen species (ROS) may be involved in the cascade of events leading to apoptosis. In the present investigation, we directly studied the kinetic generation of ROS in NO-treated neuronal cells by flow cytometry using 2', 7'-dichloro-fluorescein diacetate and dihydrorhodamine 123 as redox-sensitive fluorescence probes. The results indicated that exposure of cerebellar granule cells to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) induced oxidative stress, which was characterized by the accumulation of cytosolic and mitochondrial ROS, the increase in the extracellular hydrogen peroxide level, and the formation of lipid peroxidation products. SNAP treatment also induced apoptotic cell death as confirmed by the formation of cytosolic mono- and oligonucleosomes. Pretreating cells with the novel antioxidant EPC-K1 effectively prevented oxidative stress induced by SNAP, and attenuated cells from apoptosis.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H₂O₂-induced viability loss, which specifically evokes an autophagic response. Exposed to H₂O₂, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

Curcumin induces autophagy to protect vascular endothelial cell survival from oxidative stress damage

Our study first proposed that curcumin could protect human endothelial cells from the damage caused by oxidative stress via autophagy. Furthermore, our results revealed that curcumin causes some novel cellular mechanisms that promote autophagy as a protective effect. Pretreatment with curcumin remarkably improves the survival of human umbilical vein endothelial cells (HUVECs) from H 2O 2-induced viability loss, which specifically evokes an autophagic response. Exposed to H 2O 2, curcumin-treated HUVECs upregulate the level of microtubule-associated protein 1 light chain 3-II (LC3-II), the number of autophagosomes, and the degradation of p62. We show that this compound promotes BECN1 expression and inhibits the phosphatidylinositol 3-kinase (PtdIns3K)-AKT-mechanistic target of rapamycin (MTOR) signaling pathway. Curcumin can also reverse FOXO1 (a mediator of autophagy) nuclear localization along with causing an elevated level of cytoplasmic acetylation of FOXO1 and the interaction of acetylated FOXO1 and ATG7, under the circumstance of oxidative stress. Additionally, knockdown of FOXO1 by shRNA inhibits not only the protective effects that curcumin induced, but the autophagic process, from the quantity of LC3-II to the expression of RAB7. These results suggest that curcumin induces autophagy, indicating that curcumin has the potential for use as an autophagic-related antioxidant for prevention and treatment of oxidative stress. These data uncover a brand new protective mechanism involving FOXO1 as having a critical role in regulating autophagy in HUVECs, and suggest a novel role for curcumin in inducing a beneficial form of autophagy in HUVECs, which may be a potential multitargeted therapeutic avenue for the treatment of oxidative stress-related cardiovascular diseases.     

Increased oxidative stress induces apoptosis in human cystic fibrosis cells

Oxidative stress results in deleterious cell function in pathologies associated with inflammation. Here, we investigated the generation of superoxide anion as well as the anti-oxidant defense systems related to the isoforms of superoxide dismutases (SOD) in cystic fibrosis (CF) cells. Pro-apoptotic agents induced apoptosis in CF but not in control cells that was reduced by treatment with SOD mimetic. These effects were associated with increased superoxide anion production, sensitive to the inhibition of IκB-α phosphorylation, in pancreatic but not tracheal CF cells, and reduced upon inhibition of either mitochondrial complex I or NADPH oxidase. CF cells exhibited reduced expression, but not activity, of both Mn-SOD and Cu/Zn-SOD when compared to control cells. Although, expression of EC-SOD was similar in normal and CF cells, its activity was reduced in CF cells. We provide evidence that high levels of oxidative stress are associated with increased apoptosis in CFTR-mutated cells, the sources being different depending on the cell type. These observations underscore a reduced anti-oxidant defense mechanism, at least in part, via diminished EC-SOD activity and regulation of Cu/Zn-SOD and Mn-SOD expressions. These data point to new therapeutic possibilities in targeting anti-oxidant pathways to reduce oxidative stress and apoptosis in CF cells.     

Cadmium exposure induces vascular injury due to endothelial oxidative stress: the role of local angiotensin II and COX-2

Cadmium is an environmental pollutant that is closely linked with cardiovascular diseases, such as atherosclerosis and hypertension. Moreover, cadmium can induce an increase in oxidative stress. One of the main sites affected by oxidative stress is the aorta, which consequently develops atherosclerosis. However, there are few reports demonstrating aortic effects induced by small concentrations of cadmium that are similar to those found in the blood resulting from occupational exposure. Furthermore, several studies have reported on chronic cadmium exposure, and the results of these studies may have been influenced by the secondary effects induced by this metal, such as hypertension. Therefore, we investigated the effects of acute cadmium exposure on the vascular reactivity to phenylephrine of aortic rings isolated from male Wistar rats. Cadmium increased phenylephrine reactivity without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage or incubation with L-NAME shifted the phenylephrine concentration–response curves leftward in arteries incubated with or without cadmium, but the curves were shifted to a lesser degree after cadmium incubation. Enalapril, losartan, the nonselective COX inhibitor indomethacin, the TXA(2) synthase inhibitor furegrelate, the selective COX-2 inhibitor NS 398, the TP receptor antagonist SQ 29.548, the EP1 receptor antagonist SC 19.220, superoxide dismutase, and the NADPH oxidase inhibitor apocynin partially reverted the cadmium-induced effects on the reactivity to phenylephrine. Cadmium exposure increased vasoconstrictor activity by reducing NO bioavailability owing to the increased production of ROS by NADPH oxidase. The results of the tested cadmium concentration, which is below the reference values, suggest that acute cadmium exposure may induce vascular injury through endothelial oxidative stress. These data contribute to the evidence indicating that cadmium is a high risk to public health.     

Maslinic acid protects vascular smooth muscle cells from oxidative stress through Akt/Nrf2/HO-1 pathway

Maslinic acid (MA) is a natural triterpenoid widely distributed in edible and medicinal plants and has been demonstrated to possess bioactivity. However, its effect on vascular smooth muscle cells (VSMC) has not been explored yet. In this study, we found that heme oxygenase-1 (HO-1) expression was increased in VSMCs treated with MA. Furthermore, MA was found to induce Akt activation in a dose- and time-dependent manner. Wortmannin suppression of Akt was able to abolish HO-1 upregulation in VSMCs, suggesting the requirement of Akt activation for MA effect on HO-1. Further investigation indicated that Akt activation resulted in the elevated expression of Nrf2, a HO-1 promoter, in MA-treated VMSCs. Finally, we found that MA was able to protect VSMCs from oxidative stress induced by H₂O₂. Blocking the activation of Akt/Nrf2/HO-1 was able to compromise the protective effect of MA on VSMCs. Collectively, we provided evidence that MA protected VMSCs from oxidative stress through Akt/Nrf2/HO-1 pathway.     

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.     

Selenite induces apoptosis in sarcomatoid malignant mesothelioma cells through oxidative stress

Malignant mesothelioma cells differentiate into sarcomatoid or epithelioid phenotypes. The sarcomatoid cell type is more resistant to chemotherapy and gives a worse prognosis. We have investigated whether selenite alone and in combination with doxorubicin induced apoptosis in variously differentiated mesothelioma cells. Selenite in concentrations that could potentially be administered to patients strongly inhibited the growth of the sarcomatoid mesothelioma cells (IC50 = 7.5 microM), whereas epithelioid cells were more sensitive to doxorubicin. Benign mesothelial cells remained largely unaffected. Selenite potentiated doxorubicin treatment. Apoptosis was the dominating mode of cell death. The toxicity of selenite was mediated by oxidative stress. Furthermore the activity of the thioredoxin system was directly dependent on the concentration of selenite. This offers a possible mechanism of action of selenite treatment. Our findings suggest that selenite is a promising new drug for the treatment of malignant mesothelioma.     

Continuous exposure to l-arginine induces oxidative stress and physiological tolerance in cultured human endothelial cells

The therapeutic benefits of L-arginine (ARG) supplementation in humans, often clearly observed in short-term studies, are not evident after long-term use. The mechanisms for the development of ARG tolerance are not known and cannot be readily examined in humans. We have developed a sensitive in vitro model using a low glucose/low arginine culture medium to study the mechanisms of ARG action and tolerance using two different human endothelial cells, i.e., Ea.hy926 and human umbilical venous endothelial cells. Cultured cells were incubated with different concentrations of ARG and other agents to monitor their effects on endothelial nitric oxide synthase (eNOS) expression and function, as well as glucose and superoxide (O2(·-) ) accumulation. Short-term (2 h) exposure to at least 50 μM ARG moderately increased eNOS activity and intracellular glucose (p < 0.05), with no change in eNOS mRNA or protein expression. In contrast, 7-day continuous ARG exposure suppressed eNOS expression and activity. This was accompanied by increase in glucose and O2(·-) accumulation. Co-incubation with 100 μM ascorbic acid, 300 U/ml polyethylene glycol-superoxide dismutase (PEG-SOD), 100 μM L-lysine or 30 μM 5-chloro-2-(N-2,5-dichlorobenenesulfonamido)-benzoxazole (a fructose-1,6-bisphosphatase inhibitor) prevented the occurrence of cellular ARG tolerance. Short-term co-incubation of ARG with PEG-SOD improved cellular nitrite accumulation without altering cellular ARG uptake. These studies suggest that ARG-induced oxidative stress may be a primary causative factor for the development of cellular ARG tolerance.     

LR-90 prevents methylglyoxal-induced oxidative stress and apoptosis in human endothelial cells

Methylglyoxal (MGO) is a highly reactive dicarbonyl compound known to induce cellular injury and cytoxicity, including apoptosis in vascular cells. Vascular endothelial cell apoptosis has been implicated in the pathophysiology and progression of atherosclerosis. We investigated whether the advanced glycation end-product inhibitor LR-90 could prevent MGO-induced apoptosis in human umbilical vascular endothelial cells (HUVECs). HUVECs were pre-treated with LR-90 and then stimulated with MGO. Cell morphology, cytotoxicity and apoptosis were evaluated by light microscopy, MTT assay, and Annexin V-FITC and propidium iodide double staining, respectively. Levels of Bax, Bcl-2, cytochrome c, mitogen-activated protein kinases (MAPKs) and caspase activities were assessed by Western blotting. Reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. LR-90 dose-dependently prevented MGO-associated HUVEC cytotoxicity and apoptotic biochemical changes such as loss of MMP, increased Bax/Bcl-2 protein ratio, mitochondrial cytochrome c release and activation of caspase-3 and 9. Additionally, LR-90 blocked intracellular ROS formation and MAPK (p44/p42, p38, JNK) activation, though the latter seem to be not directly involved in MGO-induced HUVEC apoptosis. LR-90 prevents MGO-induced HUVEC apoptosis by inhibiting ROS and associated mitochondrial-dependent apoptotic signaling cascades, suggesting that LR-90 possess cytoprotective ability which could be beneficial in prevention of diabetic related-atherosclerosis.     

BRG1 interacts with Nrf2 to selectively mediate HO-1 induction in response to oxidative stress

NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.     

氧化应激诱发大脑皮质神经元凋亡的体外实验研究

既往研究表明 ,神经细胞死亡容易坏死而较少发生凋亡 ,1994年 ,Ｒａｔａｎ等培养神经细胞发现有大量神经元发生凋亡 ,但其凋亡机制尚不清楚。本实验采用氧化应激方法作用于原代培养的新生大鼠大脑皮质神经元 ,并进行凋亡指标的检测 ,以对其凋亡机制进行探讨。1　材料与方法(1)大鼠大脑皮质神经元的原代培养　取新生 1ｄ的ＳＤ大鼠 ,在无菌条件下分离皮质 ,置于盛有Ｈａｎｋ’ｓ液 ＤＭＥＭ (1∶1)小烧杯中 ,将组织剪碎并过滤至离心管中 ,加入 0 .2 5 %胰酶 2ｍｌ消化 10ｍｉｎ ,弃去胰酶溶液 ,将ＤＭＥＭ液 胎牛血清 (4∶1)加入离心管中 ,…