Reactive Oxygen Species Production In Endothelial Cells Research Articles

Spatial Mechano-Signaling Regulation of GTPases through Non-Degradative Ubiquitination.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1)toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

Selenium speciation determines the angiogenesis effect through regulating selenoproteins to trigger ROS-mediated cell apoptosis and cell cycle arrest

Tumor angiogenesis is closely related to tumor development, immune escape, and drug resistance. Therefore, the development of effective anti-tumor angiogenesis drugs is of great research significance. Although the current clinical angiogenesis inhibitors have achieved certain efficacy, they also pose the problems of limited and short duration of efficacy, drug resistance, and intrinsic toxicity. Anti-tumor angiogenesis strategies targeting endothelial cells (ECs) have attracted widespread attention in the development of highly effective and low toxicity anti-angiogenesis inhibitors. Studies have verified that the trace element selenium (Se) can inhibit tumor growth by inhibiting tumor angiogenesis through different mechanisms. Nevertheless, it is unclear whether Se speciation has different effects on anti-tumor angiogenesis. Herein, we found that Se exhibited effective anti-angiogenic activity, and its mechanisms of activity were determined by its chemical speciation. Organic Se can significantly inhibit tumor angiogenesis by targeting thioredoxin reductase (TrxR) to trigger cell apoptosis and cell cycle arrest and by increasing reactive oxygen species (ROS) production in ECs. Inorganic Se can induce cell cycle arrest and increase ROS production in ECs, showing promising anti-angiogenic effects. Se nanoparticles (SeNPs) slightly inhibit tumor angiogenesis by inducing apoptosis and cell cycle arrest and by increasing the production of ROS. In summary, this study elucidates the anti-angiogenic activity of Se speciation control with a view to providing a scientific reference for the design and development of novel Se-based highly effective and low toxicity angiogenesis inhibitors.

Endothelial Dysfunction: Is There a Hyperglycemia-Induced Imbalance of NOX and NOS?

NADPH oxidases (NOX) are enzyme complexes that have received much attention as key molecules in the development of vascular dysfunction. NOX have the primary function of generating reactive oxygen species (ROS), and are considered the main source of ROS production in endothelial cells. The endothelium is a thin monolayer that lines the inner surface of blood vessels, acting as a secretory organ to maintain homeostasis of blood flow. The enzymatic production of nitric oxide (NO) by endothelial NO synthase (eNOS) is critical in mediating endothelial function, and oxidative stress can cause dysregulation of eNOS and endothelial dysfunction. Insulin is a stimulus for increases in blood flow and endothelium-dependent vasodilation. However, cardiovascular disease and type 2 diabetes are characterized by poor control of the endothelial cell redox environment, with a shift toward overproduction of ROS by NOX. Studies in models of type 2 diabetes demonstrate that aberrant NOX activation contributes to uncoupling of eNOS and endothelial dysfunction. It is well-established that endothelial dysfunction precedes the onset of cardiovascular disease, therefore NOX are important molecular links between type 2 diabetes and vascular complications. The aim of the current review is to describe the normal, healthy physiological mechanisms involved in endothelial function, and highlight the central role of NOX in mediating endothelial dysfunction when glucose homeostasis is impaired.

BRG1 regulates NOX gene transcription in endothelial cells and contributes to cardiac ischemia-reperfusion injury

Excessive accumulation of reactive oxygen species (ROS) is considered a major culprit for a host of cardiovascular diseases. In vascular endothelial cells, ROS production is mediated by NAPDH oxidases (NOX). In the present study we investigated the role of the chromatin remodeling protein BRG1 in NOX trans-activation as well as its implication in cardiac ischemia-reperfusion injury. We report that in response to hypoxia-reoxygenation (HR) BRG1 was recruited to the NOX promoter regions in both immortalized endothelial cells and primary microvascular endothelial cells. BRG1 knockdown attenuated the induction of NOX genes by HR stimulation. Suppression of NOX trans-activation by BRG1 silencing was paralleled by the loss of active histone modifications (acetylation of histones H3 and H4) and the re-appearance of repressive histone modification (dimethylation of histone H3K9) surrounding the NOX promoter. Of interest, the H3K9 demethylase KDM3A bound to the NOX promoters with kinetics similar to BRG1 and interacted with BRG1 to activate NOX transcription. KDM3A depletion ameliorated NOX induction and ROS production in endothelial cells exposed to HR. Finally, mice with endothelial-specific deletion of BRG1 were protected from cardiac ischemia-reperfusion injury. In conclusion, our data suggest that BRG1 may link epigenetic activation of NOX transcription in endothelial cells to cardiac ischemia reperfusion injury.

ClC-3 promotes angiotensin II-induced reactive oxygen species production in endothelial cells by facilitating Nox2 NADPH oxidase complex formation.

Recent evidence suggests that ClC-3, a member of the ClC family of Cl- channels or Cl-/H+ antiporters, plays a critical role in NADPH oxidase-derived reactive oxygen species (ROS) generation. However, the underling mechanisms remain unclear. In this study we investigated the effects and mechanisms of ClC-3 on NADPH oxidase activation and ROS generation in endothelial cells. Treatment with angiotensin II (Ang II, 1 μmol/L) significantly elevated ClC-3 expression in cultured human umbilical vein endothelial cells (HUVECs). Furthermore, Ang II treatment increased ROS production and NADPH oxidase activity, an effect that could be significantly inhibited by knockdown of ClC-3, and further enhanced by overexpression of ClC-3. SA-β-galactosidase staining showed that ClC-3 silencing abolished Ang II-induced HUVEC senescence, whereas ClC-3 overexpression caused the opposite effects. We further showed that Ang II treatment increased the translocation of p47phox and p67phox from the cytosol to membrane, accompanied by elevated Nox2 and p22phox expression, which was significantly attenuated by knockdown of ClC-3 and potentiated by overexpression of ClC-3. Moreover, overexpression of ClC-3 increased Ang II-induced phosphorylation of p47phox and p38 MAPK in HUVECs. Pretreatment with a p38 inhibitor SB203580 abolished ClC-3 overexpression-induced increase in p47phox phosphorylation, as well as NADPH oxidase activity and ROS generation. Our results demonstrate that ClC-3 acts as a positive regulator of Ang II-induced NADPH oxidase activation and ROS production in endothelial cells, possibly via promoting both Nox2/p22phox expression and p38 MAPK-dependent p47phox/p67phox membrane translocation, then increasing Nox2 NADPH oxidase complex formation.

2,3′,4,4′,5-Pentachlorobiphenyl impairs insulin-induced NO production partly through excessive ROS production in endothelial cells

Polychlorinated biphenyls (PCBs) have been reported to be associated with increased risk to hypertension, atherosclerosis, cardiovascular disease, etc. 2,3′,4,4′,5-Pentachlorobiphenyl, known as PCB-118, is a member of coplanar PCBs which renders their structure similar to polychlorinated dibenzo-p-dioxins (PCDDs) and has dioxin-like activity. In our current study, we investigated the effect of PCB-118 exposure on nitric oxide (NO) production and the underlying mechanisms in vitro. Exposure of PCB-118 impaired insulin-induced NO production and endothelial nitric oxide synthase (eNOS) activity in human umbilical vein endothelial cells (HUVECs) with no significant effect on cell viability. Furthermore, PCB-118 treatment induced oxidative stress. In addition, scavenging of reactive oxygen species (ROS) by 10 μM N-acetyl-l-cysteine (NAC) partly rescued impaired insulin-induced eNOS activities and NO productions induced by PCB-118 in HUVECs. Taken together, these results indicate that PCB-118 mediates lower eNOS activity and impairs insulin-induced NO production partly through excessive ROS production in endothelial cells.

RETRACTED: Effect of thioredoxin-interacting protein on Wnt/β-catenin signaling pathway and diabetic myocardial infarction.

To explore the regulatory role of thioredoxin-interacting protein (TXNIP) in Wnt/β-catenin signaling pathway and therefore to elucidate its function in diabetic myocardial infarction. Diabetic myocardial infarction models were generated in mice. The expression levels of TXNIP and β-catenin and level of reactive oxygen species (ROS) were determined and compared with those in control group. Human umbilical vein endothelial cells were treated with high-concentration glucose and/or silencing TXNIP and/or H2O2. After 24h, expression levels of TXNIP, β-catenin and its downstream protein Cyclin D1, and C-myc gene were determined by real-time PCR, Western blot and immunofluorescence method. The cell proliferation and ROS production capability in different groups were determined by methyl thiazolyl tetrazolium assay. Compared with control group, hyperglycemia significantly up-regulated TXNIP expression and ROS level in the myocardium and endothelial cells of myocardial infarction area, whereas the β-catenin expression was down-regulated, and the difference was statistically significant (P<0.05). In comparison with Human umbilical vein endothelial cells in the control group, high glucose level increased the levels of TXNIP expression and ROS level in cells, but reduced cell proliferation as well as migration capability and expression levels of β-catenin, Cyclin D1 and C-myc; the difference was statistically significant (P<0.05). However, this trend can be partially reversed by silencing TXNIP. Diabetic myocardial ischemia could up-regulate levels of TXNIP expression and ROS production in endothelial cells of myocardial infarction area. The regulation effect of TXNIP on β-catenin was partially achieved by changing ROS levels.

NADPH OXIDASE 2, 4 AND MITOCHONDRIA-DERIVED REACTIVE OXYGEN SPECIES CONTRIBUTE TO ANGIOPOIETIN-1 INDUCED ANGIOGENESIS

The NOX proteins are reactive oxygen species (ROS) generating enzymes and together with Mitochondrial-derived ROS, they play an important role in pathological as well as physiological processes of the vasculature. Recent evidences indicate that ROS generated by NOX and the mitochondria may play important roles in growth factors downstream signaling. Our own study have pointed out that Angiopoietin-1 (Ang-1) induced ROS production is important for Ang-1/Tie-2 receptor signaling in endothelial cells, however, we did not identify the molecular sources of ROS generation in this respect. In this study, we have identified the origins of Ang-1 induced ROS production in endothelial cells and their involvement in Ang-1 induced angiogenesis. The most abundant isoforms of NOX in human umbilical endothelial cells were knocked down using adenoviruses expressing shRNA against NOX2 and NOX4. Mitochondrial-derived ROS were inhibited using SS31 peptide or Mito-Tempol. Cellular ROS levels were measured upon Ang-1 treatment using Amplex Red and Mitochondrial ROS level were measured with MitoSox. To evaluate the involvement of NOX or Mitochondrial-derived ROS in Ang-1 induced angiogenesis we evaluated cell survival by cell count and Caspase-3 cleavage, tubulogenesis by matrigel assay and migration by scratch wound assay. Activation level of the MAPK and PI3K/AKT pathways downstream of Ang-1/Tie-2 were measured by immunoblotting. Ang-1treatment leads to NOX-derived and Mitochondrial ROS production. NOX4 and the mitochondrial ROS are required for Ang-1 induced ROS production. NOX4 knockdown and Mitochondrial ROS blockage lead to impaired Ang-1 induced migration. NOX2 minimally contributes to Ang-1 induced ROS production and appears to be required for Ang-1 induced cell survival. In addition, NOX4 and mitochondrial-derived ROS are required for optimal activation of p38 and inactivation of ERK and AKT. On the other hand NOX2 appears to be required for optimal AKT activation and ERK and p38 inactivation. We conclude that NOX4 and mitochondrial ROS are required for Ang-1 induced migration through p38 activation. We also concluded that NOX2-derived ROS regulates Ang-1 induced cell survival by activation of AKT. This study provides new insight into cellular signaling mechanism downstream of Ang-1/Tie-2 signaling axis.

Abstract 452: Hyperglycemia and Oxidative Stress Impair Fluid Shear Stress-mediated Endothelial Nitric Oxide Synthase Phosphorylation and Activation via Disrupting Adherens Junctions

Endothelial dysfunction, characterized by a decrease of nitric oxide (NO) bioavailability in the vessel wall, plays a crucial role in the pathogenesis of atherosclerosis. Oxidative stress due to increased reactive oxygen species (ROS) is implicated in endothelial dysfunction associated with diabetes. However the molecular mechanisms by which oxidative stress causes endothelial dysfunction remain incompletely understood. Blood flow, which generates fluid shear stress acting on endothelium, is the most potent physiological stimulus for NO production through endothelial NO synthase (eNOS) activation. Here we report that hyperglycemia and oxidative stress impairs fluid shear stress signal transduction and eNOS activation in endothelium. We found that the exposure of endothelial cells (ECs) to ROS generators such as menadione and xanthine/xanthine oxidase inhibited laminar flow-mediated Akt and eNOS phosphorylation and activation in human endothelial cells. Moreover, ECs pre-exposed to high glucose that generates ROS production in ECs, failed to respond to laminar flow for Akt/eNOS phosphorylation and activation. Consequently NO production from ECs in response to laminar flow was attenuated by high glucose treatment. Mechanistically, we observed that hyperglycemia and oxidative stress altered endothelial adherens junction integrity, manifested by the alternation of the localization of cell-cell junction molecules vascular endothelial cadherin (VE-cadherin) and beta-catenin. Silencing VE-cadherin or beta-catenin with small interference RNA also inhibited laminar flow-mediated signaling for eNOS activation, which mimics the effects of oxidative stress, suggesting that cell-cell junction integrity is critical for fluid shear stress signal transduction and eNOS activation. Collectively, our results demonstrate that hyperglycemia and oxidative stress impair laminar flow-mediated eNOS activation through the alternation of endothelial junction integrity and fluid shear stress signal transduction. Our findings suggest a novel mechanism whereby oxidative stress induces diabetes-associated endothelial dysfunction.

Effects of 5-fluorouracil on morphology, cell cycle, proliferation, apoptosis, autophagy and ROS production in endothelial cells and cardiomyocytes.

Antimetabolites are a class of effective anticancer drugs interfering in essential biochemical processes. 5-Fluorouracil (5-FU) and its prodrug Capecitabine are widely used in the treatment of several solid tumors (gastro-intestinal, gynecological, head and neck, breast carcinomas). Therapy with fluoropyrimidines is associated with a wide range of adverse effects, including diarrhea, dehydration, abdominal pain, nausea, stomatitis, and hand-foot syndrome. Among the 5-FU side effects, increasing attention is given to cardiovascular toxicities induced at different levels and intensities. Since the mechanisms related to 5-FU-induced cardiotoxicity are still unclear, we examined the effects of 5-FU on primary cell cultures of human cardiomyocytes and endothelial cells, which represent two key components of the cardiovascular system. We analyzed at the cellular and molecular level 5-FU effects on cell proliferation, cell cycle, survival and induction of apoptosis, in an experimental cardioncology approach. We observed autophagic features at the ultrastructural and molecular levels, in particular in 5-FU exposed cardiomyocytes. Reactive oxygen species (ROS) elevation characterized the endothelial response. These responses were prevented by a ROS scavenger. We found induction of a senescent phenotype on both cell types treated with 5-FU. In vivo, in a xenograft model of colon cancer, we showed that 5-FU treatment induced ultrastructural changes in the endothelium of various organs. Taken together, our data suggest that 5-FU can affect, both at the cellular and molecular levels, two key cell types of the cardiovascular system, potentially explaining some manifestations of 5-FU-induced cardiovascular toxicity.

The 18-kDa Translocator Protein Inhibits Vascular Cell Adhesion Molecule-1 Expression via Inhibition of Mitochondrial Reactive Oxygen Species.

Translocator protein 18 kDa (TSPO) is a mitochondrial outer membrane protein and is abundantly expressed in a variety of organ and tissues. To date, the functional role of TSPO on vascular endothelial cell activation has yet to be fully elucidated. In the present study, the phorbol 12-myristate 13-acetate (PMA, 250 nM), an activator of protein kinase C (PKC), was used to induce vascular endothelial activation. Adenoviral TSPO overexpression (10–100 MOI) inhibited PMA-induced vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) expression in a dose dependent manner. PMA-induced VCAM-1 expressions were inhibited by Mito-TEMPO (0.1–0.5 μM), a specific mitochondrial antioxidants, and cyclosporin A (1–5 μM), a mitochondrial permeability transition pore inhibitor, implying on an important role of mitochondrial reactive oxygen species (ROS) on the endothelial activation. Moreover, adenoviral TSPO overexpression inhibited mitochondrial ROS production and manganese superoxide dismutase expression. On contrasts, gene silencing of TSPO with siRNA increased PMA-induced VCAM-1 expression and mitochondrial ROS production. Midazolam (1–50 μM), TSPO ligands, inhibited PMA-induced VCAM-1 and mitochondrial ROS production in endothelial cells. These results suggest that mitochondrial TSPO can inhibit PMA-induced endothelial inflammation via suppression of VCAM-1 and mitochondrial ROS production in endothelial cells.

Inhibition of calpain reduces oxidative stress and attenuates endothelial dysfunction in diabetes.

AimsThe present study was to investigate the role of calpain in reactive oxygen species (ROS) production in endothelial cells and endothelium-dependent vascular dysfunction under experimental conditions of diabetes.Methods and resultsExposure to high glucose activated calpain, induced apoptosis and reduced nitric oxide (NO) production without changing eNOS protein expression, its phosphorylation and dimers formation in primary human umbilical vein endothelial cells (HUVECs). These effects of high glucose correlated with intracellular ROS production and mitochondrial superoxide generation. Selectively scavenging mitochondrial superoxide increased NO production in high glucose-stimulated HUVECs. Inhibition of calpain using over-expression of calpastatin or pharmacological calpain inhibitor prevented high glucose-induced ROS production, mitochondrial superoxide generation and apoptosis, which were concurrent with an elevation of NO production in HUVECs. In mouse models of streptozotocin-induced type-1 diabetes and OVE26 type-1 diabetic mice, calpain activation correlated with an increase in ROS production and peroxynitrite formation in aortas. Transgenic over-expression of calpastatin reduced ROS production and peroxynitrite formation in diabetic mice. In parallel, diabetes-induced reduction of endothelium-dependent relaxation in aortic ring was reversed by over-expression of calpastatin in mouse models of diabetes. However, the protective effect of calpastatin on endothelium-dependent relaxation was abrogated by eNOS deletion in diabetic mice.ConclusionsThis study suggests that calpain may play a role in vascular endothelial cell ROS production and endothelium-dependent dysfunction in diabetes. Thus, calpain may be an important therapeutic target to overcome diabetes-induced vascular dysfunction.

Mitochondrial Dysfunction, Oxidative Stress and Diabetic Cardiovascular Disorders

The prevalence of diabetes has been rapidly increasing in world-wide countries. The dominant cause of mortality in diabetic patients is cardiovascular complications. Mechanism for the susceptibility of diabetic patients to cardiovascular disorders remains unclear. Elevated oxidative stress was detected in diabetic patients or in animal models. Mitochondria are one of major sources of reactive oxygen species (ROS) in cells. Mitochondrial dysfunction and DNA mutations have been detected in diabetic patients. Diabetes-associated metabolic disorders, including hyperglycemia, hypertriglyceridemia, hypercholesterolemia, hypoalphalipoproteinemia, and increased levels of advanced glycation end products, glycated and oxidized lipoproteins, are associated with oxidative stress. Glycated or oxidized low density lipoproteins (LDL) impair the activities of mitochondrial respiratory chain complex enzymes in vascular endothelial cells (EC). Dysfunction in mitochondrial respiration may increase the formation of ROS in mitochondria. NADPH oxidase (NOX) generates ROS in cytosol. Increased NOX activity was detected in diabetic patients. Glycated and oxidized LDL increase the expression of NOX and ROS production in EC. Diabetes-associated metabolic disorders may lead to mitochondrial dysfunction, NOX activation and excess ROS production, which results in oxidative stress and promotes cardiovascular disorders in diabetic patients. Statins, metformin and anthocyanidins may help to attenuate oxidative stress in vasculature induced by diabetes-associated metabolic disorders.

PP074. Characteristic changes in endothelial function in uterine artery by administration of no synthase inhibitor during period of placental formation

Preeclampsia is characterized as an increased peripheral vascular resistance. It is found that the administration of the NOS inhibitor l-NAME at early pregnancy developed hypertension in rats similar to preeclampsia. Furthermore, we found action of endothelial NO was reduced due to possibly reactive oxygen species (ROS) in preeclamptic women. We investigated whether this treatment modulates NO and ROS production in uterine artery endothelial cells at late pregnancy in rats. l-NAME was continuously administered during the 8-14th day of gestation in Wistar rats. Blood pressure was meassured by use of tail-cuff method. The uterine artery was obtained at the 20th day of gestation. Acetylcholine (ACh)-induced endothelial NO production was estimated from DAF-2 fluorescence. Furthermore, ACh-induced endothelial cell ROS production was estimated from CM-H2 DCFDA by fluorescence imaging system. Blood pressure was higher and birth body weight was smaller in l-NAME-treated rats than in saline-treated rats. Acetylcholine (ACh)-induced endothelial NO production was higher, while ACh-induced endothelial cell ROS production was similar, in l-NAME-treated rats when compared with those in control rats. It is suggested that the increased endothelial NO production in the uterine artery seen by a short term administration of l-NAME to rats during early pregnancy may function to protect the development of hypertension.

Peroxiredoxin 6 Phosphorylation and Subsequent Phospholipase A2 Activity Are Required for Agonist-mediated Activation of NADPH Oxidase in Mouse Pulmonary Microvascular Endothelium and Alveolar Macrophages

Peroxiredoxin 6 (Prdx6), a bifunctional enzyme with glutathione peroxidase and phospholipase A2 (PLA(2)) activities, participates in the activation of NADPH oxidase 2 (NOX2) in neutrophils, but the mechanism for this effect is not known. We now demonstrate that Prdx6 is required for agonist-induced NOX2 activation in pulmonary microvascular endothelial cells (PMVEC) and that the effect requires the PLA(2) activity of Prdx6. Generation of reactive oxygen species (ROS) in response to angiotensin II (Ang II) or phorbol 12-myristate 13-acetate was markedly reduced in perfused lungs and isolated PMVEC from Prdx6 null mice. Rac1 and p47(phox), cytosolic components of NOX2, translocated to the endothelial cell membrane after Ang II treatment in wild-type but not Prdx6 null PMVEC. MJ33, an inhibitor of Prdx6 PLA(2) activity, blocked agonist-induced PLA(2) activity and ROS generation in PMVEC by >80%, whereas inhibitors of other PLA(2)s were ineffective. Transfection of Prx6 null cells with wild-type and C47S mutant Prdx6, but not with mutants of the PLA(2) active site (S32A, H26A, and D140A), "rescued" Ang II-induced PLA(2) activity and ROS generation. Ang II treatment of wild-type cells resulted in phosphorylation of Prdx6 and its subsequent translocation from the cytosol to the cell membrane. Phosphorylation as well as PLA(2) activity and ROS generation were markedly reduced by the MAPK inhibitor, U0126. Thus, agonist-induced MAPK activation leads to Prdx6 phosphorylation and translocation to the cell membrane, where its PLA(2) activity facilitates assembly of the NOX2 complex and activation of the oxidase.

Leptin Signalling Coordinates Lipid Oxidation with Thermogenesis and Defence Against Oxidative Stress

[See original article on page 955]

Role of Reactive Oxygen Species in Tumor Necrosis Factor-alpha Induced Endothelial Dysfunction

Endothelial cell injury and dysfunction are the major triggers of pathophysiological processes leading to cardiovascular disease. Endothelial dysfunction (ED) has been implicated in atherosclerosis, hypertension, coronary artery disease, vascular complications of diabetes, chronic renal failure, insulin resistance and hypercholesterolemia. Although now recognized as a class of physiological second messengers, reactive oxygen species (ROS) are important mediators in cellular injury, specifically, as a factor in endothelial cell damage. Uncontrolled ROS production and/or decreased antioxidant activity results in a deleterious state referred to as 'oxidative stress'. A candidate factor in causing ROS production in endothelial cells is tumor necrosis factor alpha (TNF-α), a pleiotropic inflammatory cytokine. TNF-α has been shown to both be secreted by endothelial cells and to induce intracellular ROS formation. These observations provide a potential mechanism by which TNF-α may activate and injure endothelial cells resulting in ED. In this review, we focus on the relationship between intracellular ROS formation and ED in endothelial cells or blood vessels exposed to TNF-α to provide insight into the role of this important cytokine in cardiovascular disease.

An Oral Adsorbent, AST-120 Protects Against the Progression of Oxidative Stress by Reducing the Accumulation of Indoxyl Sulfate in the Systemic Circulation in Renal Failure

The effect of AST-120, an oral adsorbent, on oxidative stress in the systemic circulation in chronic renal failure (CRF) was examined and the potential role of indoxyl sulfate (IS), an uremic toxin adsorbed by AST-120, in inducing the formation of reactive oxygen species (ROS) in the vascular system was studied, in vitro and in vivo. The level of oxidized albumin, a marker for oxidative stress in the systemic circulation was determined by HPLC, as previously reported. The mRNA levels of TGF-beta (1) and Oat1 were measured by quantitative RT-PCR. The IS induced ROS generation in cultured human umbilical vein endothelial cells (HUVECs) was estimated using a fluorescence microplate reader. An increase in the ratio of oxidized to unoxidized albumin was determined using 5/6 nephrectomized rats (CRF rats) compared to a control group. The ratio was significantly reduced in the group that received AST-120 of 4 weeks, suggesting that AST-120 inhibits oxidative stress in CRF. An anti-oxidative effect of AST-120 was also observed in CRF rats with a similar renal function. The ratio of oxidized albumin was correlated with serum IS levels in vivo. The same relationship was also observed in CRF rats with the continued administration of IS. In addition, IS dramatically increased the generation of ROS in both a dose- and time- dependent manner in HUVEC, suggesting that accumulated IS may play an important role in enhancing intravascular oxidative stress. We propose that AST-120 reduces IS concentrations in the blood that induces ROS production in endothelial cells, thereby inhibiting the subsequent occurrence of oxidative stress in the systemic circulation in renal failure.

Isoliquiritigenin inhibits IκB kinase activity and ROS generation to block TNF-α induced expression of cell adhesion molecules on human endothelial cells

Isoliquiritigenin (ILTG) is a flavonoid with chalcone structure (4,2′,4′-trihydroxychalcone), an active component present in plants like Glycyrrhiza and Dalbergia which showed various biological activities including anti-inflammatory, anti-carcinogenic and antihistamic. As very little is known in regard to the underlying mechanism involved in explaining the various activities of the compound, we carried out a detailed study on the effect of ILTG on the expression of cell adhesion molecules on human primary endothelial cells. We demonstrate here that ILTG inhibits TNF-α induced adhesion of neutrophils to endothelial monolayer by blocking the expression of ICAM-1, VCAM-1 and E-selectin. Since NF-κB is a major transcription factor involved in the transcriptional regulation of cell adhesion molecules, thus we studied the status of NF-κB activation in ILTG treated endothelial cells. We demonstrate that ILTG inhibits the translocation and activation of nuclear factor-κB (NF-κB) by blocking the phosphorylation and subsequent degradation of IκBα. As oxidative stress is also known to regulate the activation of NF-κB to modulate TNF-α signaling cascade, we tested the effect of ILTG on reactive oxygen species (ROS). We found that it inhibits TNF-α induced ROS production in endothelial cells. These results have important implications for using ILTG or its derivatives towards the development of effective anti-inflammatory molecules.

Thrombin stimulates calcium‐dependent production of mitochondrial ROS in pulmonary microvascular endothelial cells

Vascular cells respond to various stimuli via enhanced production of reactive oxygen species (ROS). In endothelial cells, proteins that produce ROS include the flavoenzyme NADPH oxidase of cell membranes and the mitochondrial electron transport chain. Thrombin, an enzyme linked to atherosclerosis and restenosis injury, can trigger cellular oxidative stress, although the mechanism for its effects in endothelial cells is unclear. To determine the mechanism by which thrombin evokes endothelial cell signaling, murine pulmonary microvascular endothelial cells (MPMVECs) were simultaneously loaded with the calcium indicator dye Fluo-4/AM and the mitochondrial membrane potential indicator tetramethylrhodamine, ethyl ester, perchlorate (TMRE) and visualized by confocal microscopy. Thrombin stimulated a large calcium release from the intracellular stores that preceded mitochondrial membrane potential loss. Subsequently, mitochondrial superoxide production was enhanced as evidenced by an increase in fluorescence of the mitochondrial superoxide-sensitive fluorophore, MitoSOX Red. Thrombin treatment also increased nuclear HE fluorescence, an indicator of cellular superoxide production, and was unaffected in the prescence of the NADPH oxidase inhibitor Apocynin and the anion channel blocker DIDS. In contrast, Apocynin and DIDS ablated the HE fluorescence increase observed in response to Angiotensin II, a known stimulator of NADPH oxidase. Thus, thrombin stimulates ROS production in endothelial cells primarily via mitochondria, and may play an important role in the vascular response in areas adjacent to injury. [HL-60290 & HL-07748]