Reactive Oxygen Species Generation In Endothelial Cells Research Articles

Canagliflozin inhibits inflammasome activation in diabetic endothelial cells – Revealing a novel calcium-dependent anti-inflammatory effect of canagliflozin on human diabetic endothelial cells

BackgroundCanagliflozin (CANA) shows anti-inflammatory and anti-oxidative effects on endothelial cells (ECs). In diabetes mellitus (DM), excessive reactive oxygen species (ROS) generation, increased intracellular calcium (Ca2+) and enhanced extracellular signal regulated kinase (ERK) 1/2 phosphorylation are crucial precursors for inflammasome activation. We hypothesized that: (1) CANA prevents the TNF-α triggered ROS generation in ECs from diabetic donors and in turn suppresses the inflammasome activation; and (2) the anti-inflammatory effect of CANA is mediated via intracellular Ca2+ and ERK1/2. MethodsHuman coronary artery endothelial cells from donors with DM (D-HCAECs) were pre-incubated with either CANA or vehicle for 2 h before exposure to 50 ng/ml TNF-α for 2–48 h. NAC was applied to scavenge ROS, BAPTA-AM to chelate intracellular Ca2+, and PD 98059 to inhibit the activation of ERK1/2. Live cell imaging was performed at 6 h to measure ROS and intracellular Ca2+. At 48 h, ELISA and infra-red western blot were applied to detect IL-1β, NLRP3, pro-caspase-1 and ASC. Results10 µM CANA significantly reduced TNF-α related ROS generation, IL-1β production and NLRP3 expression (P all <0.05), but NAC did not alter the inflammasome activation (P > 0.05). CANA and BAPTA both prevented intracellular Ca2+ increase in cells exposed to TNF-α (P both <0.05). Moreover, BAPTA and PD 98059 significantly reduced the TNF-α triggered IL-1β production as well as NLRP3 and pro-caspase-1 expression (P all <0.05). ConclusionCANA suppresses inflammasome activation by inhibition of (1) intracellular Ca2+ and (2) ERK1/2 phosphorylation, but not by ROS reduction.

Lysophosphatidylcholine induces oxidative stress in human endothelial cells via NOX5 activation - implications in atherosclerosis.

The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. Human aortic endothelial cells (HAEC) were stimulated with LPC (10-5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10-7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

Butyrate protects endothelial function through PPARδ/miR-181b signaling

Reports of the beneficial roles of butyrate in cardiovascular diseases, such as atherosclerosis and ischemic stroke, are becoming increasingly abundant. However, the mechanisms of its bioactivities remain largely unknown. In this study, we explored the effects of butyrate on endothelial dysfunction and its potential underlying mechanism. In our study, ApoE-/- mice were fed with high-fat diet (HFD) for ten weeks to produce atherosclerosis models and concurrently treated with or without sodium butyrate daily. Thoracic aortas were subsequently isolated from C57BL/6 wild-type (WT), PPARδ-/-, endothelial-specific PPARδ wild-type (EC-specific PPARδ WT) and endothelial-specific PPARδ knockout (EC-specific PPARδ KO) mice were stimulated with interleukin (IL)-1β with or without butyrate ex vivo. Our results demonstrated that butyrate treatment rescued the impaired endothelium-dependent relaxations (EDRs) in thoracic aortas of HFD-fed ApoE-/- mice. Butyrate also rescued impaired EDRs in IL-1β-treated thoracic aorta ring ex vivo. Global and endothelial-specific knockout of PPARδ eliminated the protective effects of butyrate against IL-1β-induced impairment to EDRs. Butyrate abolished IL-1β-induced reactive oxygen species (ROS) production in endothelial cells while the inhibitory effect was incapacitated by genetic deletion of PPARδ or pharmacological inhibition of PPARδ. IL-1β increased NADPH oxidase 2 (NOX2) mRNA and protein expressions in endothelial cells, which were prevented by butyrate treatment, and the effects of butyrate were blunted following pharmacological inhibition of PPARδ. Importantly, butyrate treatment upregulated the miR-181b expression in atherosclerotic aortas and IL-1β-treated endothelial cells. Moreover, transfection of endothelial cells with miR-181b inhibitor abolished the suppressive effects of butyrate on NOX2 expressions and ROS generation in endothelial cells. To conclude, butyrate prevents endothelial dysfunction in atherosclerosis by reducing endothelial NOX2 expression and ROS production via the PPARδ/miR-181b pathway.

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.

Cysteamine prevents vascular leakage through inhibiting transglutaminase in diabetic retina.

Cysteamine (an aminothiol), which is derived from coenzyme A degradation and metabolized into taurine, has beneficial effects against cystinosis and neurodegenerative diseases; however, its role in diabetic complications is unknown. Thus, we sought to determine the preventive effect of cysteamine against hyperglycemia-induced vascular leakage in the retinas of diabetic mice. Cysteamine and ethanolamine, the sulfhydryl group-free cysteamine analogue, inhibited vascular endothelial growth factor (VEGF)-induced stress fiber formation and vascular endothelial (VE)-cadherin disruption in endothelial cells, which play a critical role in modulating endothelial permeability. Intravitreal injection of the amine compounds prevented hyperglycemia-induced vascular leakage in the retinas of streptozotocin-induced diabetic mice. We then investigated the potential roles of reactive oxygen species (ROS) and transglutaminase (TGase) in the cysteamine prevention of VEGF-induced vascular leakage. Cysteamine, but not ethanolamine, inhibited VEGF-induced ROS generation in endothelial cells and diabetic retinas. In contrast, VEGF-induced TGase activation was prevented by both cysteamine and ethanolamine. Our findings suggest that cysteamine protects against vascular leakage through inhibiting VEGF-induced TGase activation rather than ROS generation in diabetic retinas.

NADPH Oxidase Activation and Endothelial Nitric Oxide Synthase Uncoupling Contribute to Endothelial Dysfunction in Antiphospholipid Syndrome

Introduction: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI). APLA cause endothelial dysfunction, though the underlying mechanisms have not been clearly delineated.Methods: Endothelial cells (EC) were incubated with β2GPI and either control antibodies or anti-β2GPI antibodies affinity-purified from sera of patients with APS, in the absence or presence of diapocynin, an NADPH oxidase (NOX) inhibitor, or siRNA against NOX1, NOX2, or NOX4. Generation of reactive oxygen species (ROS) in EC and conditioned medium were measured using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or chemiluminescent substrate. NOX1, NOX2, NOX4, 3-nitrotyrosine, and thioredoxin reductase 1 (TrxR1) expression were analyzed by western blot. eNOS monomer and dimer were detected using cold (4°C) SDS-PAGE and immunoblot. Immunoprecipitation of TrxR1 was performed using protein A/G agarose. EC activation was assessed by measuring expression of E-selectin.Results: Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated ROS generation in EC, as well as the release of ROS into conditioned medium. Expression of NOX2, but not NOX1 or NOX4, was significantly increased in EC exposed to anti-β2GPI antibodies, but not control IgG. Preteatment of endothelial cells with diapocynin, a NOX inhibitor, or siRNA against NOX2 and NOX4, but not NOX1, inhibited endothelial cell activation by anti-β2GPI antibodies. Furthermore, anti-β2GPI antibody-treated EC generated more peroxynitrite, as determined by 3-nitrotyrosine expression. Treatment of EC with anti-β2GPI antibodies increased eNOS monomer/dimer ratio, suggesting eNOS uncoupling. Compared to control human IgG, the TrxR1 immunoprecipitate from EC treated with β2GPI and anti-β2GPI antibodies contained more 3-nitrotyrosine level, suggesting TrxR1 tyrosine residue modification by nitration and possible loss of function.Conclusions: β2GPI and anti-β2GPI antibodies stimulate ROS generation in EC, with release into the conditioned medium. The impairment of EC activation by diapocynin, NOX2 siRNA, or NOX4 siRNA suggests that NOX mediate EC activation by anti-β2GPI antibodies. Anti-β2GPI antibodies induce nitrative stress in EC, which might be explained by eNOS uncoupling induced by these antibodies. The nitration of TrxR1, an enzyme that prevents eNOS uncoupling, may result in the inactivation of TrxR1 and contribute to eNOS uncoupling. Taken together, these studies provide preliminary evidence of the contribution of NOX activation and eNOS uncoupling in mediating oxidative and nitrative stress and induction of EC dysfunction by APLA. DisclosuresMcCrae:Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees.

NADPH Oxidase Mediates Antiphospholipid Antibody-Induced Endothelial Cell Activation

Introduction: Antiphospholipid Syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI), an abundant plasma phospholipid binding protein. APLA/anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner, though the underlying mechanisms are not well defined.Objective: To define the role of NOX1 in the generation of ROS and activation of endothelial cells by anti-β2GPI antibodies.Methods: Endothelial cells were incubated with β2GPI and either control or affinity-purified anti-β2GPI antibodies in the absence or presence of diphenyleneiodonium (DPI), an NADPH oxidase (NOX) inhibitor. Generation of reactive oxygen species (ROS) in treated cells and conditioned medium were measured by using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or luminescent substrate. NOX mRNA and protein expression were assessed using quantitative PCR and immunoblot. Endothelial cell activation was measured by increased expression of E-selectin.Results: Incubation of endothelial cells with β2GPI and anti-β2GPI antibodies stimulated ROS generation in endothelial cells, as well as the release of ROS into conditioned medium. The expression of NOX1 mRNA and protein levels were significantly increased in endothelial cells exposed to anti-β2GPI antibodies, but not control IgG (Figure 1). The ability of β2GPI and anti-β2GPI antibodies to induce endothelial cell E-selectin mRNA expression was blocked by pretreatment of cells with DPI (Figure 2), suggesting that ROS is required for downstream events underlying endothelial cell activation.Conclusions: Endothelial cells exposed to β2GPI and anti-β2GPI antibodies generate ROS, which is subsequently released into the conditioned medium. NOX1 appears to be essential for ROS generation. The impairment of endothelial cell activation by DPI suggests that NOX is also essential for endothelial cell activation by anti-β2GPI antibodies. We hypothesize that production of ROS by NOX plays a central role in APLA-induced endothelial dysfunction.Acknowledgment: This work was supported by an ASH Bridge Grant [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.

Acute Exposure to Diesel Exhaust Impairs Nitric Oxide–Mediated Endothelial Vasomotor Function by Increasing Endothelial Oxidative Stress

Exposure to diesel exhaust was recently identified as an important cardiovascular risk factor, but whether it impairs nitric oxide (NO)-mediated endothelial function and increases production of reactive oxygen species (ROS) in endothelial cells is not known. We tested these hypotheses in a randomized, controlled, crossover study in healthy male volunteers exposed to ambient and polluted air (n=12). The effects of skin microvascular hyperemic provocative tests, including local heating and iontophoresis of acetylcholine and sodium nitroprusside, were assessed using a laser Doppler imager. Before local heating, skin was pretreated by iontophoresis of either a specific NO-synthase inhibitor (L-N-arginine-methyl-ester) or a saline solution (Control). ROS production was measured by chemiluminescence using the lucigenin technique in human umbilical vein endothelial cells preincubated with serum from 5 of the subjects. Exposure to diesel exhaust reduced acetylcholine-induced vasodilation (P<0.01) but did not affect vasodilation with sodium nitroprusside. Moreover, the acetylcholine/sodium nitroprusside vasodilation ratio decreased from 1.51 ± 0.1 to 1.06 ± 0.07 (P<0.01) and was correlated to inhaled particulate matter 2.5 (r=-0.55; P<0.01). NO-mediated skin thermal vasodilatation decreased from 466 ± 264% to 29 ± 123% (P<0.05). ROS production was increased after polluted air exposure (P<0.01) and was correlated with the total amount of inhaled particulate matter <2.5 μm (PM2.5). In healthy subjects, acute experimental exposure to diesel exhaust impaired NO-mediated endothelial vasomotor function and promoted ROS generation in endothelial cells. Increased PM2.5 inhalation enhances microvascular dysfunction and ROS production.

Protective Effects of Low-Frequency Magnetic Fields on Cardiomyocytes from Ischemia Reperfusion InjuryviaROS and NO/ONOO−

Background. Cardiac ischemia reperfusion (I/R) injury is associated with overproduction of reactive oxygen species (ROS). Low frequency pulse magnetic fields (LFMFs) have been reported to decrease ROS generation in endothelial cells. Whether LFMFs could assert protective effects on myocardial from I/R injury via ROS regulation remains unclear. Methods. To simulate in vivo cardiac I/R injury, neonatal rat cardiomyocytes were subjected to hypoxia reoxygenation (H/R) with or without exposure to LFMFs. Cell viability, apoptosis index, ROS generation (including O2 − and ONOO−), and NO production were measured in control, H/R, and H/R + LFMF groups, respectively. Results. H/R injury resulted in cardiomyocytes apoptosis and decreased cell viability, whereas exposure to LFMFs before or after H/R injury significantly inhibited apoptosis and improved cell viability (P < 0.05). LFMFs treatment could suppress ROS (including O2 − and ONOO−) generation induced by H/R injury, combined with decreased NADPH oxidase activity. In addition, LFMFs elevated NO production and enhanced NO/ONOO− balance in cardiomyocytes, and this protective effect was via the phosphorylation of endothelial nitric oxide synthase (eNOS). Conclusion. LFMFs could protect myocardium against I/R injury via regulating ROS generation and NO/ONOO− balance. LFMFs treatment might serve as a promising strategy for cardiac I/R injury.

MJ33, an inhibitor of the phospholipase A2 activity of peroxiredoxin 6, reduces reactive oxygen species production in a model of endotoxin induced lung inflammation

NADPH oxidases (NOX) are a major source of enzymatically generated reactive oxygen species (ROS) in mammalian cells; of these, Nox2 activation is thought to play a role in the injury associated with lung inflammation. We reported that Nox2 activation and ROS production occurs through peroxiredoxin 6 (Prdx6) and its subsequent PLA2 activity. Thus blocking Prdx6 PLA2 activity can be a strategy to inhibit ROS generation during inflammation. Here we used MJ33, a potent Prdx6 PLA2 inhibitor and evaluated ROS generation in endothelial cells in vitro and in mouse lungs in vivo post LPS treatment. LPS was either added to endothelial cells in culture or injected i.v. or i.t in mice, while MJ33 was added directly to cell cultures or administered i.t. at doses between 1–10 mole % in liposomes. ROS generation in endothelial cells (monitored by H2DCF fluorescence) showed a 3‐ fold increase after LPS treatment as compared to controls; however addition of MJ33 reduced ROS levels to control values. Lungs from mice treated with LPS showed an almost 10 fold higher rate of ROS production (as monitored by H2O2 specific dye, amplex red) which was abolished by pretreatment with MJ33. Administration of MJ33 in LPS treated mice reduced ROS levels to values as seen with Prdx6 null and Nox2 null lungs indicating that Prdx6 PLA2 activity is critical to LPS induced ROS production and that MJ33 treatment may provide a favorable outcome during inflammation.

Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes

Objectives: Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice. Methods: Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured. Results: High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice. Conclusions: This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.

Adenine Nucleotide Translocator as a Regulator of Mitochondrial Function: Implication in the Pathogenesis of Metabolic Syndrome

Mitochondria play key roles in energy production and intracellular reactive oxygen species (ROS) generation. Lines of evidence have shown that mitochondrial dysfunction contributes to the development of metabolic syndrome. The causes of mitochondrial dysfunction are complex, but overnutrition and sedentary living are among the best known causes of mitochondrial dysfunction. ATP synthesized in the mitochondria is exchanged for cytosolic ADP by adenine nucleotide translocator (ANT) to provide a continuous supply of ADP to mitochondria. We recently found that ANT function is essential for peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α)'s action on endothelial cells. PGC-1α is a transcriptional coactivator of nuclear receptors, playing an important role in fatty acid oxidation and mitochondrial biogenesis. Recent studies have shown that PGC-1α decreases intracellular ROS generation by increasing the expression of antioxidant genes. In our study, PGC-1α reduced cell apoptosis and ROS generation in endothelial cells by increasing ATP/ADP translocase activity of ANT and ANT1 expression. Here we review the role of ANT in maintaining proper mitochondrial function, and possible role of ANT dysfunction in the pathogenesis of metabolic syndrome.

Impairment of mitochondrial respiratory chain activity in aortic endothelial cells induced by glycated low-density lipoprotein

Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD +/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.

Role of LOX‐1 in monocyte adhesion‐triggered redox, Akt/eNOS and Ca2+ signaling pathways in endothelial cells

This study was conducted to examine the role of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in monocyte adhesion-induced redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in endothelial cells (ECs). LOX-1 was blocked by an antibody-neutralizing LOX-1 TS92 or small interfering RNA. In cultured human aortic ECs, monocyte adhesion activated Rac1 and p47(phox), and increased NADPH oxidase activity and reactive oxygen species (ROS) generation within 30 min and NF-kappaB phosphorylation within 1 h, resulting in redox-sensitive gene expression. Akt and eNOS phosphorylation was induced 15 min after adding monocytes and returned to control level after 30 min, whereas NO production was not altered by monocyte adhesion. Blockade of LOX-1 blunted the monocyte adhesion-triggered redox-sensitive signaling pathway and Akt/eNOS phosphorylation in ECs. Both endothelial intracellular Ca2+ mobilization and Ca2+ influx caused by monocyte attachment were markedly attenuated by pretreatment of ECs with TS92. This suggests that LOX-1 is involved in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of oxidized low-density lipoprotein (ox-LDL). Furthermore, blockade of Ca2+ inhibited monocyte adhesion-triggered Rac1 and p47(phox) activation and ROS generation in ECs, whereas Ca2+ signaling was suppressed by blockade of NADPH oxidase and ROS generation. Finally, TS92 blocked the monocyte adhesion to ECs stimulated with or without tumor necrosis factor-alpha or ox-LDL. We provide evidence that LOX-1 plays a role in redox-sensitive, Akt/eNOS and Ca2+ signaling pathways in monocyte adhesion to ECs independent of the ox-LDL-LOX-1 axis.

Activation of endothelial cells after exposure to ambient ultrafine particles: The role of NADPH oxidase

Several studies have shown that ultrafine particles (UFPs) may pass from the lungs to the circulation because of their very small diameter, and induce lung oxidative stress with a resultant increase in lung epithelial permeability. The direct effects of UFPs on vascular endothelium remain unknown. We hypothesized that exposure to UFPs leads to endothelial cell O 2 − generation via NADPH oxidase and results in activation of endothelial cells. Our results showed that UFPs, at a non-toxic dose, induced reactive oxygen species (ROS) generation in mouse pulmonary microvascular endothelial cells (MPMVEC) that was inhibited by pre-treatment with the ROS scavengers or inhibitors, but not with the mitochondrial inhibitor, rotenone. UFP-induced ROS generation in MPMVEC was abolished by p67 phox siRNA transfection and UFPs did not cause ROS generation in MPMVEC isolated from gp91 phox knock-out mice. UFP-induced ROS generation in endothelial cells was also determined in vivo by using a perfused lung model with imaging. Moreover, Western blot and immunofluorescence staining results showed that MPMVEC treated with UFPs resulted in the translocation of cytosolic proteins of NADPH oxidase, p47 phox, p67 phox and rac 1, to the plasma membrane. These results demonstrate that NADPH oxidase in the pulmonary endothelium is involved in ROS generation following exposure to UFPs. To investigate the activation of endothelial cells by UFP-induced oxidative stress, we determined the activation of the mitogen-activated protein kinases (MAPKs) in MPMVEC. Our results showed that exposure of MPMVEC to UFPs caused increased phosphorylation of p38 and ERK1/2 MAPKs that was blocked by pre-treatment with DPI or p67 phox siRNA. Exposure of MPMVEC obtained from gp91 phox knock-out mice to UFPs did not cause increased phosphorylation of p38 and ERK1/2 MAPKs. These findings confirm that UFPs can cause endothelial cells to generate ROS directly via activation of NADPH oxidase. UFP-induced ROS lead to activation of MAPKs through induced phosphorylation of p38 and ERK1/2 MAPKs that may further result in endothelial dysfunction through production of cytokines such as IL-6. Our results suggest that endothelial oxidative stress may be an important mechanism for PM-induced cardiovascular effects.

Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells

Fabry disease, an X-linked systemic vasculopathy, is caused by a deficiency of α-galactosidase A resulting in globotriaosylceramide (Gb 3) storage in cells. The pathogenic role of Gb 3 in the disease is not known. Based on previous work, we tested the hypothesis that accumulation of Gb 3 in the vascular endothelium of Fabry disease is associated with increased production of reactive oxygen species (ROS) and increased expression of cell adhesion molecules. Gb 3-loading resulted in increased intracellular ROS production in cultured vascular endothelial cells in a dose-dependent manner. Increased Gb 3 also induced expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. Reduction of endogenous Gb 3 by treatment of the cells with an inhibitor of glycosphingolipid synthase or α-galactosidase A led to decreased expression of adhesion molecules. Plasma from Fabry patients significantly increased ROS generation in endothelial cells when compared with plasma from non-Fabry controls. This effect was not influenced by reduction of intracellular Gb 3. This study provided direct evidence that excess intracellular Gb 3 induces oxidative stress and up-regulates the expression of cellular adhesion molecules in vascular endothelial cells. In addition, other factors in patient’s plasma may also contribute to oxidative stress in Fabry vascular endothelial cells.

Reversal of oxidative stress in endothelial cells by controlled release of adiponectin

Hyperglycemia causes endothelial dysfunction due to its effect on increasing reactive oxygen species (ROS). Adiponectin (Adp) has been reported to suppress hyperglycemia-associated ROS generation. It was hypothesized that administering globular adiponectin (gAdp) via injectable biodegradable thermosensitive triblock copolymer might effectively reduce ROS generation in endothelial cells. In this study, gAdp was incorporated into and released from the polymer gel. The released gAdp was further investigated by comparing it with the intact gAdp with regard to the efficiency in reducing ROS and activating cAMP. The released gAdp effectively suppressed excess ROS production in the in vitro endothelial cell culture model under high-glucose condition via cAMP/PKA pathway. These data provide a rationale for developing controlled release dosage form of gAdp as a therapeutic tool for oxidative stress-related pathology in patients with diabetes.

Aspirin-triggered lipoxin A4 blocks reactive oxygen species generation in endothelial cells: A novel antioxidative mechanism

Lipoxins and their aspirin-triggered carbon-15 epimers have emerged as mediators of key events in endogenous anti-inflammation and resolution. However, the implication of these novel lipid mediators on cardiovascular diseases such as hypertension, atherosclerosis, and heart failure has not been investigated. One of the major features shared by these pathological conditions is the increased production of reactive oxygen species (ROS) generated by vascular NAD(P)H oxidase activation. In this study, we have examined whether an aspirin-triggered lipoxin A (4) analog (ATL-1) modulates ROS generation in endothelial cells (EC). Pre-treatment of EC with ATL-1 (1 - 100 nM) completely blocked ROS production triggered by different agents, as assessed by dihydrorhodamine 123 and hydroethidine. Furthermore, ATL-1 inhibited the phosphorylation and translocation of the cytosplamic NAD(P)H oxidase subunit p47 (phox) to the cell membrane as well as NAD(P)H oxidase activity. Western blot and immunofluorescence microscopy analyses showed that ATL-1 (100 nM) impaired the redox-sensitive activation of the transcriptional factor NF- kappaB, a critical step in several events associated to vascular pathologies. These results demonstrate that ATL-1 suppresses NAD(P)H oxidase-mediated ROS generation in EC, strongly indicating that lipoxins may play a protective role against the development and progression of cardiovascular diseases.

Sphingosylphosphorylcholine induces apoptosis of endothelial cells through reactive oxygen species‐mediated activation of ERK

Sphingosylphosphorylcholine (SPC) produces reactive oxygen species (ROS) in MS1 pancreatic islet endothelial cells. In the present study, we explored the physiological significance of the SPC-induced ROS generation in endothelial cells. SPC induced cell death of MS1 cells at higher than 10 microM concentration through a caspase-3-dependent pathway. SPC treatment induced sustained activation of an extracellular signal-regulated kinase (ERK), in contrast to transient activation of ERK in response to platelet-derived growth factor (PDGF)-BB, which stimulated proliferation of MS1 cells. Both the SPC-induced cell death and ERK activation were abolished by pretreatment of the cells with the MEK inhibitor U0126 or by overexpression of a dominant negative mutant of MEK1 (DN-MEK1). Pretreatment of the cells with N-acetylcysteine, an antioxidant, completely prevented the SPC-induced ROS generation, apoptosis, and ERK activation, whereas the ROS generation was not abrogated by treatment with U0126. Consistent with these results, SPC induced cell death of human umbilical vein endothelial cells (HUVECs) through ROS-mediated activation of ERK. These results suggest that the SPC-induced generation of ROS plays a crucial role in the cell death of endothelial cells through ERK-dependent pathway.