Mediated Reactive Oxygen Species Generation Research Articles

Cryptotanshinone, an orally bioactive herbal compound from Danshen, attenuates atherosclerosis in apolipoprotein E-deficient mice: role of lectin-like oxidized LDL receptor-1 (LOX-1).

Cryptotanshinone (CTS) is a major bioactive diterpenoid isolated from Danshen, an eminent medicinal herb that is used to treat cardiovascular disorders in Asian medicine. However, it is not known whether CTS can prevent experimental atherosclerosis. The present study was designed to investigate the protective effects of CTS on atherosclerosis and its molecular mechanisms of action. Apolipoprotein E-deficient (ApoE(-/-)) mice, fed an atherogenic diet, were dosed daily with CTS (15, 45 mg kg(-1) day(-1)) by oral gavage. In vitro studies were carried out in oxidized LDL (oxLDL)-stimulated HUVECs treated with or without CTS. CTS significantly attenuated atherosclerotic plaque formation and enhanced plaque stability in ApoE(-/-) mice by inhibiting the expression of lectin-like oxLDL receptor-1 (LOX-1) and MMP-9, as well as inhibiting reactive oxygen species (ROS) generation and NF-κB activation. CTS treatment significantly decreased the levels of serum pro-inflammatory mediators without altering the serum lipid profile. In vitro, CTS decreased oxLDL-induced LOX-1 mRNA and protein expression and, thereby, inhibited LOX-1-mediated adhesion of monocytes to HUVECs, by reducing the expression of adhesion molecules (intracellular adhesion molecule 1 and vascular cellular adhesion molecule 1). Furthermore, CTS inhibited NADPH oxidase subunit 4 (NOX4)-mediated ROS generation and consequent activation of NF-κB in HUVECs. CTS was shown to have anti-atherosclerotic activity, which was mediated through inhibition of the LOX-1-mediated signalling pathway. This suggests that CTS is a vasculoprotective drug that has potential therapeutic value for the clinical treatment of atherosclerotic cardiovascular diseases.

RhoA and Rac1 Gtpases Differentially Regulate Agonist-Receptor Mediated ROS Generation in Platelets

Agonist induced generation of reactive oxygen species (ROS) including superoxide anion (O-2) and hydrogen peroxide (H2O2) enhance platelet aggregation and hence the risk of thrombosis. Although diverse biochemical reactions contribute to ROS generation, NADPH oxidases (NOX) have emerged as critical sources of agonist induced ROS generation in platelets. Previous studies have shown that small GTPases Rac1 and RhoA are involved in NOX activation. Rac GTPase activates NOX by directly binding to NOX as well as by interacting with p67phox to promote its binding to NOX (Physiol Rev 87: 245–313, 2007), whereas RhoA triggers ROS generation via the ROCK/p38MAPK cascade mediated phosphorylation of p47phox, a critical component of the NOX complex, (Exp Mol Med 37:575-87, 2005). To date, however, the roles of Rac1 and RhoA in platelet ROS production remain unclear. This study was conducted to define the contributions of Rac1- and RhoA- signaling to ROS generation and platelet function. ROS generation was quantified by flow cytometry in dcf-da (10 µM) loaded washed platelets. Thrombin has been shown to generate ROS in human platelets (Blood 106: 2757-2760, 2005). In this study we confirmed that platelets stimulated with thrombin generate ROS in a time- and a concentration- dependent manner. Addition of thrombin to human platelets pre-treated with NSC23766, a Rac-specific inhibitor, or murine platelets with Rac1 gene deletion, produced significantly less ROS than the matching control samples. Further, Phox-I, a pharmacologic inhibitor of Rac-p67phox interaction (Chem Biol 19: 228-24, 2012), potently suppressed thrombin induced ROS production, indicating that a Rac1-p67phox signaling axis is involved in thrombin mediated ROS production. Separately, treatment of washed human platelets with a RhoA specific inhibitor, Rhosin (Chem Biol 19:699-710, 2012) resulted in: (a) inhibition of the U46619, a stable analog of TXA2, induced activation of RhoA, but not that of Rac1or Cdc42; (b) U46619 induced phosphorylation p38MAPK and p47phox; and (c) U46619 or thrombin induced ROS generation. We further investigated the role of RhoA/ROCK/p38MAPK in ROS production by using platelets from RhoA-/- mice, Y27632 (a ROCK inhibitor) and SB203580 (a p38MAPK inhibitor). RhoA-/- platelets or human platelets treated with Y27632 or SB203580 exhibited significantly diminished ROS generation in response to thrombin. Next, we investigated the physiological effects of Rhosin on platelet activation. A pre-incubation of washed human platelets with Rhosin inhibited U46619 or thrombin induced platelet shape change, release of P-selectin, secretion of ATP and aggregation. The anti-platelet effects of Rhosin were reversible as washing of platelets after incubation with Rhosin abolished the inhibitory effect of Rhosin on platelet aggregation. These results suggest that (a) RhoA signaling, through ROCK/MAPK/p47phox activation, leads to ROS generation and platelet activation in conjunction with or independent of the RhoA/ROCK mediated phosphorylation of MLC, and (b) Rac1 and RhoA differentially regulate platelet ROS generation by directly binding to NOX, promoting binding of p67phox to NOX and by phosphorylation of p47phox, respectively. DisclosuresNo relevant conflicts of interest to declare.

Homer1 knockdown protects dopamine neurons through regulating calcium homeostasis in an in vitro model of Parkinson's disease

Homer1 protein is an important scaffold protein at postsynaptic density and has been demonstrated to play a central role in calcium signaling in the central nervous system. The aim of this study was to investigate the effects of Homer1 knockdown on MPP+ induced neuronal injury in cultured dopamine (DA) neurons. We found that down-regulating Homer1 expression with specific small interfering RNA (siRNA) significantly suppressed LDH release, reduced Propidium iodide (PI) or Hoechst staining, increased the number of tyrosine hydroxylase (TH) positive cells and DA uptake, and attenuated apoptotic and necrotic cell death after MPP+ injury. Homer1 knockdown decreased intracellular reactive oxygen species (ROS) generation through inhibition of intracellular calcium overload, but did not affect the endogenous antioxidant enzyme activities. Calcium imaging was used to examine the changes of intracellular Ca2+ concentration ([Ca2+]cyt) and Ca2+ in endoplasmic reticulum (ER) ([Ca2+]ER), and the results showed that Homer1 siRNA transfection attenuated ER Ca2+ release up to 120min after MPP+ injury. Furthermore, decrease of [Ca2+]cyt induced by Homer1 knockdown in MPP+ treated neurons was further enhanced by NMDA receptor antagonists MK-801 and AP-5, but not canonical transient receptor potential (TRPC) channel antagonist SKF-96365. l-type calcium antagonist isradipine but not nimodipine further inhibited intracellular calcium overload after MPP+ insult in Homer1 down-regulated neurons. These results suggest that Homer1 knockdown has protective effects against neuronal injury in in vitro PD model by reducing calcium overload mediated ROS generation, and this protection may be dependent at least in part on the regulatory effects on the function of calcium channels in both plasma membrane and ER.

NADPH oxidase‐4 mediated ROS generation modulates C2C12 differentiation via ERK1/2 pathway

BackgroundNADPH oxidase 4 (Nox4) enzymatic activity is a major source of intracellular reactive oxygen species (ROS) and plays an important role in balancing oxidation‐reduction within skeletal muscle cells. We hypothesize that changes in Nox4 mediated ROS generation affects the proliferation and differentiation of skeletal muscle cells. Further, ROS plays an important role in cell signaling pathways and this is dependent on the concentration of endogenous ROS in the cells.ApproachUsing mouse myogenic (C2C12) cells, Nox4 concentrations were manipulated via either plasmid mediated over‐expression or siRNA silencing resulting in increased or decreased endogenous ROS concentrations, respectively. Real time RT‐PCR and Western blot demonstrated effective modulation of Nox4 isoform and endogenous ROS production was measured using flow‐cytometry. We further quantified Nox4 protein expression and myogenin using immunofluorescence microscopy. ERK1/2 phosphorylation was determined by Western blot.ResultsModulation of Nox4 and endogenous ROS concentrations decreased the expression of myogenic differentiation markers, Myf5, Myogenin, MyoD1 and Pax7. This is associated with a decrease in cell differentiation percentage in the early stages of myogenesis. These changes are associated with altered MAPK pathway activation; specifically phosphorylation of ERK1/2. In both over‐expression and silencing of Nox4, we observed significant decreases in ERK1/2 phosphorylation.ConclusionNox4 activity determines optimum levels of ROS that are important for cellular processes including proliferation and differentiation of C2C12 myoblasts. Changes in myogenic differentiation pathways in response to Nox4 modulation are mediated via MAPK activation.Funding: USDA/NIFA award number 2010–34479‐20715 “Increasing Shelf Life of Agricultural Commodities, ID”

Stem-bark of Terminalia arjuna attenuates human monocytic (THP-1) and aortic endothelial cell activation

Ethnopharmacological relevanceTerminalia arjuna – stem bark extract is traditionally used as cardiotonic in Ayurvedic medicine. Aim of the studyThe present study was aimed to evaluate the molecular basis for cardioprotective potential of Terminalia arjuna (TA) stem bark, using cell cultures of human monocytic (THP-1) and human aortic endothelial cells (HAECs). Materials and methodsInhibitory effect of alcoholic (TAAE) and aqueous (TAWE) extracts of TA-stem bark was assessed on human 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, lipoprotein lipase (LpL) and lipid peroxidation in rat (wistar) liver and heart homogenates. The patterns of H2O2 induced reactive oxygen species (ROS) generation were observed by confocal microscopy. The activities of antioxidant enzymes and reducing power of the cells were measured in a microplate reader. Gene transcripts of proinflammatory markers in THP-1 and HAECs were assayed by real time PCR and levels of inflammatory protein markers by ELISA or flow cytometry. Phytochemical analyses of TAAE and TAWE were done using liquid chromatography, coupled to mass spectrometry (LC-MS). ResultsTAAE and TAWE inhibited the lipid peroxidation and HMG-CoA reductase but had no effect on LpL. Both the extracts attenuated H2O2 mediated ROS generation in THP-1 cells by promoting catalase (CAT), glutathione peroxidase (GPx) activities, and by sustaining cellular reducing power. TAAE was highly effective in attenuating proinflammatory gene transcripts in THP-1 cells and HAECs, whereas the response to TAWE depended on the type of transcript and cell type. Both extracts decreased the levels of typical inflammatory marker proteins, viz. LPS induced tumor necrosis factor (TNF)-α secreted by THP-1 cells and TNF-α induced cell surface adhesion molecules on HAECs, namely vascular cell adhesion molecule-1 (VCAM-1) and E-selectin. Phytochemical analyses indicated the richness in phenolic compounds and terpenes of TAAE and TAWE, while revealing variability in their metabolite profile. ConclusionOur study scientifically validates the antioxidative and antiinflammatory properties of Terminalia arjuna stem bark. The marked effects on cultured human monocytic and aortic endothelial cells (HAEC) provide the biochemical and molecular basis for therapeutic potential of TA-stem bark against cardiovascular diseases (CVD).

Wogonin Induces Reactive Oxygen Species Production and Cell Apoptosis in Human Glioma Cancer Cells

Glioma is the most common primary adult brain tumor with poor prognosis because of the ease of spreading tumor cells to other regions of the brain. Cell apoptosis is frequently targeted for developing anti-cancer drugs. In the present study, we have assessed wogonin, a flavonoid compound isolated from Scutellaria baicalensis Georgi, induced ROS generation, endoplasmic reticulum (ER) stress and cell apoptosis. Wogonin induced cell death in two different human glioma cells, such as U251 and U87 cells but not in human primary astrocytes (IC 50 > 100 μM). Wogonin-induced apoptotic cell death in glioma cells was measured by propidine iodine (PI) analysis, Tunnel assay and Annexin V staining methods. Furthermore, wogonin also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Moreover, treatment of wogonin also increased a number of signature ER stress markers glucose-regulated protein (GRP)-78, GRP-94, Calpain I, and phosphorylation of eukaryotic initiation factor-2α (eIF2α). Treatment of human glioma cells with wogonin was found to induce reactive oxygen species (ROS) generation. Wogonin induced ER stress-related protein expression and cell apoptosis was reduced by the ROS inhibitors apocynin and NAC (N-acetylcysteine). The present study provides evidence to support the fact that wogonin induces human glioma cell apoptosis mediated ROS generation, ER stress activation and cell apoptosis.

Uncovering the biological function of UQCRB, a terpestacin‐binding mitochondrial protein, implies its pro‐angiogenic activity in vitro and in vivo

Recently, we identified ubiquinol‐cytochrome c reductase binding protein (UQCRB), one of subunits of mitochondrial Complex III, as a target protein of the anti‐angiogenic natural small molecule, terpestacin. The Complex III of mitochondrial respiratory chain has been considered as a key regulator in hypoxia‐induced angiogenesis through mitochondria‐derived reactive oxygen species (ROS) involved in O2 sensing. Here, cell permeable UQCRB protein is generated using protein transduction domain (PTD), a small peptide transferring its binding complex into the cell, to reveal the biological function of UQCRB. Consequently, intracellular delivery of PTD‐UQCRB fusion protein enhances generation of mitochondrial ROS and HIF‐1α stability. Also, PTD‐UQCRB transduction induces vascular endothelial growth factor (VEGF) expression and invasion of human umbilical vascular endothelial cells (HUVECs) in vitro. Furthermore, trans‐membrane delivery of PTD‐UQCRB enhances wound healing in vivo. These results provide new insights into the function of PTD‐UQCRB in angiogenesis via mitochondria mediated ROS generation and also open new basis on application of PTD‐UQCRB as a pro‐angiogenic agent via regulating mitochondrial function. This study is supported by self‐research funding.

Elevate the ROS Level to Kill Cancer Cells during Chemotherapy

Chemotherapy and radiotherapy are an essential treatment for many cancers, sometimes in combination with cytoreductive surgery. While radiotherapy destroys cancer cells by ionizing radiation that directly damages DNA, most of the chemotherapeutic agents generate Reactive Oxygen Species (ROS) that lead to oxidative damage in various molecules of the cell [1]. However, both processes mainly induce apoptosis to kill cancer cells. Any cancer cells that are not killed by these therapies could proliferate again, resulting in the relapse of cancer. The clinical benefit of using antioxidant supplements along with chemotherapy and radiotherapy is highly debatable, and not conclusive. This is either due to incomplete investigations, lack of enough samples for suitable statistical analysis, or failure to correlate wide ranges of different parameters used in different studies [2]. However, some of the clinical studies suggest that the antioxidant supplemented group had a worst survival rate, than the group who did not use antioxidant supplements [3]. Although in some cases, use of antioxidant has fewer side effects leading to less damage to normal tissues, but with a decrease in the overall survival rate. The ROS generated by anticancer agents, although effective in killing cancer cells, also alters other cellular pathways leading to various side effects [4]. The serious side effects encountered in chemotherapy include nephrotoxicity, ototoxicity or cardiotoxicity [2]. However, it is also believed that antioxidant supplemented reduction of side effects depends, mainly on using specific anticancer drugs for certain cancers.Thus, the intriguing question is whether antioxidant should continue to be used along with chemotherapy, or should be avoided. Also, the question arises whether cancer cells should be killed completely to cure cancers, without considering side effects. Logically, complete cure of cancer by overcoming drug resistance with minimal side effects should be the first priority.A systematic search including chemotherapy and ROS generation yielded more than 13,000 publications in the PubMed database, including mainly clinical studies. Searching anticancer agents mediated ROS generation yielded 307 studies in most recent years, demonstrating an intense focus shift towards understanding the mechanism of drug-mediated cancer cell death, through ROS generation. This shift could be attributed to the use of current genomic and molecular biology tools, to help understand adverse outcomes of chemotherapy and drug resistance that are observed in decades of clinical treatments. Arguably, as most of the recent studies have pointed out, if those anticancer agents generate ROS to kill cancer cells, then why antioxidant would be used along with chemotherapy? Instead, elevation of ROS should be the most effective way to supplement anticancer drugs. In addition, it has been observed in several recent reports that a reduced ROS level is attributed to drug resistance in certain cancer cells for some anticancer drugs [1,5], and indirectly supported by other studies [6,7]. Thus, increase of ROS level along with the drug could help in overcoming drug resistance during chemotherapy.Now what are the means to elevate ROS level in the cancer cell for effective killing with chemotherapy? The simple answers are by manipulating antioxidant genes or ROS maintaining genes/enzymes, or by conjugating nanoparticles that generate ROS, or by introducing external ROS to the tumor sites. Although, several antioxidant genes are known such as SODs (superoxide dismutases), catalase, GSH (glutathione), thioredoxins and peroxiredoxins, the ROS maintaining genes (e.g. ARHGEF6, p53, APEX1 etc.), and their mechanisms are largely unknown. Nanoparticles-mediated ROS generation technologies are also being developed [8]. The most important question is how effective overall would it be to overcome drug resistance by elevating the ROS level? At this point, extensive investigations could be initiated to assess the effect of elevated level of ROS to overcome drug resistance for many anticancer drugs in different types of cancers. After successful characterization in cellular and mouse models, the results could be implemented in systematic clinical trials. Thus, in the coming years, advanced chemotherapeutic treatments could be supplemented by means of ROS elevation, rather than with antioxidants.

Quercetin suppresses cytochrome P450 mediated ROS generation and NFκB activation to inhibit the development of 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinomas

Increased production of reactive oxygen species (ROS) has long been recognized to play a pivotal role in carcinogenesis. Quercetin, a naturally occurring dietary flavonoid is known for its ROS scavenging properties. The present study was designed to investigate the chemopreventive and chemotherapeutic effects of quercetin based on cytochrome P450 (CYP) mediated ROS generation, ROS-induced cellular damage and activation of the NFκB signalling circuit during 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Administration of quercetin inhibited the development of DMBA-induced HBP carcinomas by impairing CYP-mediated ROS production via downregulation of the expression of CYP1A1 and CYP1B1, and upregulation of antioxidant defences. Attenuation of ROS generation by quercetin in turn abrogated NFκB signalling by preventing the phosphorylation and degradation of IκB, nuclear translocation of NFκB and transactivation of its target genes associated with cell proliferation and apoptosis evasion. Thus dietary flavonoids such as quercetin that can block ROS generation and inhibit the redox regulated transcription factor NFκB, by virtue of their antioxidant potential are promising candidates for future antioxidant-based anticancer regimens.

Gum resin of Boswellia serrata inhibited human monocytic (THP-1) cell activation and platelet aggregation

Ethnopharmacological relevanceStem bark gum resin extract of Boswellia serrata is traditionally used in India for its hemostatic, antiinflammatory and cardiovascular health effects and it is named as Śallakī in Ayurvedic medicine. Aim of the studyThis study was conducted to evaluate the antioxidative and antithrombotic properties of stem bark gum resin extracts of Boswellia serrata (BS). Materials and methodsThe inhibitory activity of the BSWE and BSAE on FeCl3 induced lipid peroxidation (in vitro) in rat liver and heart homogenates was measured spectrophotometrically. Their effect on H2O2 induced reactive oxygen species (ROS) generation in human monocytic (THP-1) cells was investigated by tracking intensity of a cell permeable fluorescent dye, H2DCFDA and subjecting the cell samples to confocal microscopy. Further, the effect of BSAE and BSWE on ADP-induced platelet aggregation was assessed using a multimode detection plate reader, plasma coagulation times using an automated blood coagulation analyzer and on human blood clotting factors Xa and XIa using chromogenic substrate. Phytomarker analysis of the water (BSWE) and hydroalcoholic (BSAE) extracts of BS-gum resin was done through HPLC using a standard compound AKβBA. ResultsBSAE and BSWE inhibited, to varied extents, the lipid peroxidation in liver (80%) and heart (50%) tissue homogenates of male Wistar rats. Further, BSAE (30μgdwt/mL) and BSWE (300μgdwt/mL) attenuated ≥60% of H2O2 mediated ROS generation in THP-1 cells. In case of standard compounds, ascorbate (20μgdwt/mL) and butylated hydroxytoluene (BHT) (10μgdwt/mL) completely scavenged ROS in the cells. BSAE and BSWE at 3mgdwt/mL completely inhibited ADP induced platelet aggregation and activities were comparable to 20μg/mL of heparin. The extracts also showed very high activity in prolonging coagulation time periods. Both types of extracts extended prothrombin time (PT) from ∼13 to >60s and activated partial thromboplastin time (APTT) from ∼32s to >90s. BSAE inhibited clotting factors Xa and XIa remarkably at 6μg of dwt where as BSWE did not show much effect on FXa and showed 30% inhibition on FXIa at 120μg. 10μg of heparin was required to inhibit about 30% activity of the above factors. HPLC analyses suggested that BSAE and BSWE had AKβBA of 9% (w/w) and 7.8% (w/w) respectively. ConclusionPresent study demonstrated antioxidant and antithrombotic anticoagulant activities of water and hydroalcoholic extracts of Boswellia serrata's gum resin. We suggest that BS-gum resin as a good source for lead/therapeutic compounds possessing antioxidant, antiplatelet and anticoagulant activities.

Intermittent Hypoxia Increases Endothelin‐1‐Induced Reactive Oxygen Species Generation through PKC Signaling in Small Pulmonary Arteries

Intermittent hypoxia (IH) augments endothelin-1 (ET-1) induced pulmonary vasoconstrictor reactivity through a PKCα/β and reactive oxygen species (ROS)-dependent mechanism. Although this response may contribute to IH induced pulmonary hypertension, the mechanism by which IH upregulates PKC signaling is unknown as is whether PKC and ROS signal in series to mediate this response. We hypothesized that increased pulmonary arterial PKCα/β expression and activity after IH mediate the ET-1 induced ROS generation not seen under control conditions. The PKCα/β inhibitor myr-PKC (10 μM) decreased ET-1 stimulated ROS levels as measured by dihydroethidium (DHE) fluorescence in endothelium-disrupted, pressurized pulmonary arteries (~150 μm diameter) from IH rats (3 min cycles; 5% O2, 5% CO2/air flush; 7 h/day; 4 wk) but not in arteries from sham (air cycled) rats. Although total pulmonary arterial PKCα/β mRNA or protein levels did not differ between groups, basal levels of PKCβI/II were greater in the membrane fraction of IH arteries compared to sham, consistent with elevated PKCβ activity. Therefore IH appears to augment ET-1 mediated ROS generation in pulmonary arteries through a PKCα/β signaling mechanism independent of altered PKC expression.

Early Inflammatory Reactions in Atherosclerosis Are Induced by Proline-Rich Tyrosine Kinase/Reactive Oxygen Species–Mediated Release of Tumor Necrosis Factor-α and Subsequent Activation of the p21 Cip1 /Ets-1/p300 System

Reactive oxygen species (ROS) are involved in the initial process of atherosclerosis, whereas it remains to be determined how atherogenic stimulus causes ROS-mediated proinflammatory reactions. Here, we focused on proline-rich tyrosine kinase (PYK2)-mediated ROS generation and examined how atherogenic stimulus causes early proinflammatory reactions. PYK2-deficient (knockout [KO]) (PYK2-KO) mice were crossbred with apolipoprotein E (ApoE)-deficient (PYK2-KO/ApoE-KO) mice. PYK2-KO/ApoE-KO mice and endothelial cells (EC) were used for the study. Aortic atherogenic lesions in PYK2-KO/ApoE-KO mice were markedly decreased (55% versus ApoE-KO) after 8 weeks of a Western diet. Aortic PYK2 was activated as early as 7 days after the Western diet, when inflammatory cells were not yet activated. Addition of the proatherogenic oxidized phospholipid lysophosphatidylcholine caused activation of endothelial PYK2. Lysophosphatidylcholine-activated PYK2 induced NADPH oxidase-mediated ROS generation and ROS-mediated synthesis of tumor necrosis factor-α (TNFα), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and p21Cip1/Ets-1. Neutralizing anti-TNFα antibody or knockdown of p21Cip1/Ets-1 system blocked the induction of VCAM-1 and MCP-1. PYK2 deficiency abolished these ROS-mediated proinflammatory reactions. Further analysis revealed that PYK2/ROS-mediated p21Cip1/Ets-1 activation upregulated the transcription of the MCP-1 gene in collaboration with p300 transcription coactivator. PYK2 is a key tyrosine kinase activated by high cholesterol exposure, which causes ROS-mediated TNFα release and induces TNFα-dependent expression of proinflammatory molecules via the p21Cip1/Ets-1/p300 transcription system.

Pigment epithelium-derived factor inhibits advanced glycation end products-induced retinal vascular permeability

Vascular hyperpermeability associated with retinal vascular leakage is known to occur in patients with diabetes, and contributes to endothelial barrier dysfunction. This study aimed to examine the effect of pigment epithelium-derived factor (PEDF) on advanced glycation end products (AGEs)-induced endothelial cell permeability. Cultured porcine retinal endothelial cell (PREC) was exposed to AGE-modified bovine serum albumin (AGE-BSA) and the endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the PREC monolayers. We found that AGE-BSA increased the RITC-dextran flux across a PREC monolayer and PEDF blocked the solute flux induced by AGE-BSA. In order to explore the underlying signaling mechanism of PEDF on the inhibitory effect of AGE-BSA-induced permeability, we demonstrate that PEDF could inhibit the AGE-BSA-induced permeability via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. AGE-BSA also increased the endothelial cell permeability by stimulating the reactive oxygen species (ROS) generation via NADPH oxidase activity and Akt phosphorylation at Ser473. PEDF decreased ROS generation in AGE-BSA-exposed endothelial cells by suppressing the NADPH oxidase activity via down regulating the phosphorylation of p22 PHOx at Thr147. This led to blockade of AGE-induction of PI3K/Akt activation in permeability. Furthermore, PEDF inhibited the AGE-BSA-induced permeability by increased expression of tight junction protein zona occludens-1(ZO-1), co-incident with an increase in barrier properties of endothelial monolayer. Together, our results indicate that PEDF could possibly act as potent anti-permeability molecule by targeting the PI3K/Akt signaling pathway by suppressing if NADPH oxidase mediated ROS generation and ZO-1 tight junction protein and it offers potential targets to inhibit the ocular related diseases such as diabetic retinopathy.

Abstract 1636: Anticancer drug-induced Jak2/Stat3 activation confers a survival advantage in lung cancer cells

Abstract Chemotherapy may trigger survival signaling in cancer cells and the cells may change the response to chemotherapy. Here we found chemotherapeutics-induced Jak2/Stat3 signaling activation, by which cancer cells escape from death in human lung cancer PC14PE6AS2 (AS2) and H157 cells with constitutively activated Stat3 protein. Cisplatin, paclitaxel and epirubicin are important agents in lung cancer treatment and they are reactive oxygen species (ROS)-producing agents. In AS2 and H157 cells, we found that during the paclitaxel treatment, early Jak2 activation occurred and then Stat3 tyrosine phosphorylation upregulated early. But Stat3 serine phosphorylation was not significantly increased. When AS2 and H157 cells were treated with cisplatin and epirubicin, the Jak2/Stat3 activation was also observed, which can be blocked by AG490. It is indicated that chemotherapy, such as paclitaxel, cisplatin and epirubicin, may activate Jak2/Stat3 pathway. In AS2 cells, chemotherapeutics generated ROS, and the chemotherapy-induced Jak2/Stat3 activation was only effectively inhibited by mitochondrial ROS scavengers as rotenone and antimycin. The similar finding was also observed in H157 cells. It is said that “mild” mitochondrial uncoupling provides a survival benefit in cancer cells. In AS2 and H157 cells, pretreatment with the mitochondrial uncoupler carbonylcyanide-4-trifluoromethoxy-phenylhydrazone (FCCP) inhibited paclitaxel-induced mitochondrial ROS generation, abolished paclitaxel-induced Stat3 activation in AS2 and H157 cells, and augmented paclitaxel-induced mitochondrial membrane potential decrease. Thereafter, we classified AS2 and H157 cells as “FCCP-responsive” or “less mitochondrial uncoupling” cancer cells. So that mitochondrial coupling-mediated oxidative stress modulated paclitaxel-induced Jak2/Stat3 activation in AS2 and H157 cells. Subsequently, Stat3 activation by chemotherapeutics enhanced survivin expression in AS2 and H157 cells. Pretreatment with AG490, FCCP and rotenone blocked paclitaxel-induced survivin expression in a dose-dependent manner. Pretreatment with FCCP and rotenone also made AS2 and H157 cells more sensitive to the drug. Conclusively, anti-cancer drugs-induced Jak2/Stat3 activation confers a survival advantage in lung cancer AS2 and H157 cells via mitochondrial-coupling mediated ROS generation. This is a new phenomenon of cellular response to chemotherapeutics, by which cancer cells escape from drug-induced death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1636.

Regulation of mitochondrial fission by intracellular Ca2+ in rat ventricular myocytes

Mitochondria are dynamic organelles that constantly undergo fission, fusion, and movement. Increasing evidence indicates that these dynamic changes are intricately related to mitochondrial function, suggesting that mitochondrial form and function are linked. Calcium (Ca2+) is one signal that has been shown to both regulate mitochondrial fission in various cell types and stimulate mitochondrial enzymes involved in ATP generation. However, although Ca2+ plays an important role in adult cardiac muscle cells for excitation–metabolism coupling, little is known about whether Ca2+ can regulate their mitochondrial morphology. Therefore, we tested the role of Ca2+ in regulating cardiac mitochondrial fission. We found that neonatal and adult cardiomyocyte mitochondria undergo rapid and transient fragmentation upon a thapsigargin (TG)- or KCl-induced cytosolic Ca2+ increase. The mitochondrial fission protein, DLP1, participates in this mitochondrial fragmentation, suggesting that cardiac mitochondrial fission machinery may be regulated by intracellular Ca2+ signaling. Moreover, the TG-induced fragmentation was also associated with an increase in reactive oxygen species (ROS) formation, suggesting that activation of mitochondrial fission machinery is an early event for Ca2+-mediated ROS generation in cardiac myocytes. These results suggest that Ca2+, an important regulator of muscle contraction and energy generation, also dynamically regulates mitochondrial morphology and ROS generation in cardiac myocytes.

The Adenosine A2A Receptor Modulates TRPV1 Expression and Function by Inhibiting Nerve Growth Factor (NGF) Signaling in Peripheral Neurons

NGF is implicated in sensitization of nociceptive neurons and mediation of inflammatory pain. One mechanism underlying this process is activation and induction of transient receptor potential vanilloid 1 (TRPV1) channel via generation of reactive oxygen species (ROS) and activation of p38 mitogen activated protein kinase (MAPK) pathway. Previous studies from our laboratory showed that NGF regulates the expression of the adenosine A2A receptor (A2AR) through activation of nuclear factor (NF)‐κ B via the p75 receptor. In this study, we determined whether activation of the A2AR could in turn modulate NGF signaling and TRPV1 channel expression in rat pheochromocytoma (PC12) and dorsal root ganglion (DRG) cells, known to express both of these receptors and are responsive to NGF. We observed that the A2AR agonist, CGS21680 (1 μM) reduced NGF mediated ROS generation (via the NADPH oxidase system) in both DRG and PC12 cultures and reduced p38 MAPK activation. Furthermore, CGS21680 reduced the induction of TRPV1 expression by NGF. These results suggest that the A2AR could serve as an endogenous regulator of NGF signaling and thereby modulates the thermal/inflammatory pain pathways controlled by TRPV1. Disruption of such an endogenous regulator would be expected to exacerbate the pain response by removing this inhibition of NGF signaling. This study was supported by an Excellence in Academic Medicine grant from SIU School of Medicine.

Impaired Priming and Activation of the NADPH Oxidase in Patients with TLR Signaling Defects

The NADPH oxidase of neutrophils (PMN) plays a key role in host defense by producing reactive oxygen species (ROS). Here we examine the molecular basis of priming and activation of the NADPH oxidase in PMN from patients with two types of TLR signaling defects: interleukin‐1 receptor associated kinase‐4 deficiency (IRAK4) and NF‐kB essential modulator deficiency. (NEMO) We observed Impaired superoxide generation by LPS‐primed and fMLP‐activated IRAK4 PMN that correlated with inefficient translocation of cytosolic factors: p47phox, p67phox and Rac2 and the flavo‐cytochrome subunit: gp91phox to the membrane. While IRAK4 PMN produced ≤ 5% of normal ROS levels, NEMO PMN displayed ~50% of normal ROS with normal translocation of regulatory subunits. Compared to LPS‐primed normal PMN, p47phox phosphorylation was significantly lower in IRAK4 PMN. We further observed the down‐modulation of p47phox phosphorylation and membrane translocation by phosphatidylinositol 3‐kinase (PI3K) inhibitor LY2940002 in LPS‐primed normal PMN. In addition the phosphorylated levels of signaling kinases p21‐activated kinase (Paks) (IRAK4 deficiency) and p38 (IRAK4 and NEMO deficiency) were significantly lower in PMN of TLR signaling defects. We, hereby, reveal the role of defective p38‐ PI3K‐ Rac‐Paks signaling pathway in IRAK4 deficiency patient in impairing the NADPH oxidase mediated ROS generation.Research support: NIH, NIAID

Ethanol impairs CCK-8-evoked amylase secretion through Ca 2+-mediated ROS generation in mouse pancreatic acinar cells

In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca 2+ concentration ([Ca 2+] c) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3 nM of 19.39 ± 2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1–50 mM). In contrast, incubation of cells with 50 mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca 2+] c resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca 2+. Additionally, ethanol led to an increase in fluorescence of CM-H 2DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca 2+ and in the presence of ethylene glycol-bis(2-aminoethylether)- N, N, N′, N′-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca 2+ chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and in the absence of extracellular Ca 2+, the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca 2+-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.

Mechanism of 1-Methyl-4-Phenylpyridinium-Induced Dopamine Release from PC12 Cells

The molecular mechanism of 1-methyl-4-phenylpyridinium (MPP+), a Parkinsonism-inducing neurotoxin, has been studied in PC12 cells. The cells treated with MPP+ (100 microM) induced a rapid increase in phosphorylation of tyrosine residues of several proteins, including synaptophysin, a major 38 kDa synaptic vesicle protein implicated in exocytosis. An accelerated release of dopamine by MPP+ correlated with phosphorylation of synaptophysin. Exposing the cells to MPP+ triggered reactive oxygen species (ROS) generation within 60 min of treatment and the said effect was blocked by mazindol, a dopamine uptake blocker. In addition, pretreatment with 50-100 microM of selegiline, a selective MAO-B inhibitor, significantly suppressed MPP+-mediated ROS generation. These effects of MPP+ result in the generation of ROS, which may be involved in neuronal degeneration seen in Parkinson's disease.

The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles

The effect of reactive oxygen species (ROS), produced by the mitochondrial respiratory chain, on the activity of cytochrome c oxidase and on the cardiolipin content in bovine heart submitochondrial particles (SMP) was studied. ROS were produced by treatment of succinate-respiring SMP with antimycin A. This treatment resulted in a large production of superoxide anion, measured by epinephrine method, which was blocked by superoxide dismutase (SOD). Exposure of SMP to mitochondrial mediated ROS generation, led to a marked loss of cytochrome c oxidase activity and to a parallel loss of cardiolipin content. Both these effects were completely abolished by SOD+catalase. Added cardiolipin was able to almost completely restore the ROS-induced loss of cytochrome c oxidase activity. No restoration was obtained with peroxidized cardiolipin. These results demonstrate that mitochondrial mediated ROS generation affects the activity of cytochrome c oxidase via peroxidation of cardiolipin which is needed for the optimal functioning of this enzyme complex. These results may prove useful in probing molecular mechanism of ROS-induced peroxidative damage to mitochondria which have been proposed to contribute to aging, ischemia/reperfusion and chronic degenerative diseases.