Superoxide Production In Cells Research Articles

NADPH oxidase activation contributes to native low-density lipoprotein-induced proliferation of human aortic smooth muscle cells.

Elevated plasma concentration of native low-density lipoprotein (nLDL) is associated with vascular smooth muscle cell (VSMC) activation and cardiovascular disease. We investigated the mechanisms of superoxide generation and its contribution to pathophysiological cell proliferation in response to nLDL stimulation. Lucigenin-induced chemiluminescence was used to measure nLDL-induced superoxide production in human aortic smooth muscle cells (hAoSMCs). Superoxide production was increased by nicotinamide adenine dinucleotide phosphate (NADPH) and decreased by NADPH oxidase inhibitors in nLDL-stimulated hAoSMC and hAoSMC homogenates, as well as in prepared membrane fractions. Extracellular signal-regulated kinase 1/2 (Erk1/2), protein kinase C-θ (PKCθ) and protein kinase C-β (PKCβ) were phosphorylated and maximally activated within 3 min of nLDL stimulation. Phosphorylated Erk1/2 mitogen-activated protein kinase, PKCθ and PKCβ stimulated interactions between p47phox and p22phox; these interactions were prevented by MEK and PKC inhibitors (PD98059 and calphostin C, respectively). These inhibitors decreased nLDL-dependent superoxide production and blocked translocation of p47phox to the membrane, as shown by epifluorescence imaging and cellular fractionation experiments. Proliferation assays showed that a small interfering RNA against p47phox, as well as superoxide scavenger and NADPH oxidase inhibitors, blocked nLDL-induced hAoSMC proliferation. The nLDL stimulation in deendothelialized aortic rings from C57BL/6J mice increased dihydroethidine fluorescence and induced p47phox translocation that was blocked by PD98059 or calphostin C. Isolated aortic SMCs from p47phox−/− mice (mAoSMCs) did not respond to nLDL stimulation. Furthermore, NADPH oxidase 1 (Nox1) was responsible for superoxide generation and cell proliferation in nLDL-stimulated hAoSMCs. These data demonstrated that NADPH oxidase activation contributed to cell proliferation in nLDL-stimulated hAoSMCs.

Plasma from Type 2 Diabetes Patients Increase Monocyte‐Derived Superoxide Production via ER Stress‐NADPH Oxidase Pathway

Recent evidence suggests that when human monocytes (THP-1 cells) were incubated with artificially made glucolipotoxic media to mimic the plasma composition of type 2 diabetes (T2D) patients, intracellular reactive oxygen species were increased via endoplasmic reticular (ER) stress. The extent to which these findings translate to patients with T2D remains unclear. First, we measured intracellular superoxide production (dihydroethidium fluorescence) in peripheral blood mononuclear cells from whole blood of T2D patients (N=6) and compared to healthy controls (CON; N=6). T2D patients exhibited greater basal intracellular superoxide (3.1±0.5 fold; P<0.05). Next, THP-1 cells were cultured with plasma from T2D patients or CON. The cells exposed to T2D plasma exhibited greater intracellular superoxide compared to CON (1.2±0.03 fold; P<0.05). Importantly, the increase in superoxide was eliminated by tauroursodeoxycholic acid (TUDCA; ER stress inhibitor) and gp91ds-tat (NADPH oxidase inhibitor) either alone or in combination. Lastly, when glucose (high: 25 mM or normal: 5.9 mM) alone was cultured with THP-1 cells, no increase in the monocyte-derived superoxide was found (P>0.05). Collectively, these data suggest that the ability of ER stress to induce human monocyte-derived superoxide in T2D appears to be mediated by NADPH oxidase. Moreover, hyperglycemia alone does not appear to be a primary contributor of elevated superoxide, suggesting that other potential triggers in plasma of T2D patients contribute to ER stress-induced NADPH oxidase activation in monocytes. Supported by MU CBIS pilot grant (NIH grant# P50AT006273) (SHD)

Pulmonary manifestations in adult patients with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by failure of superoxide production in phagocytic cells. The disease is characterised by recurrent infections and inflammatory events, frequently affecting the lungs. Improvement of life expectancy now allows most patients to reach adulthood. We aimed to describe the pattern of pulmonary manifestations occurring during adulthood in CGD patients. This was a retrospective study of the French national cohort of adult patients (≥16 years old) with CGD. Medical data were obtained for 67 adult patients. Pulmonary manifestations affected two-thirds of adult patients. Their incidence was significantly higher than in childhood (mean annual rate 0.22 versus 0.07, p=0.01). Infectious risk persisted despite anti-infectious prophylaxis. Invasive fungal infections were frequent (0.11 per year per patient) and asymptomatic in 37% of the cases. They often required lung biopsy for diagnosis (10 out of 30). Noninfectious respiratory events concerned 28% of adult patients, frequently associated with a concomitant fungal infection (40%). They were more frequent in patients with the X-linked form of CGD. Immune-modulator therapies were required in most cases (70%). Respiratory manifestations are major complications of CGD in adulthood. Noninfectious pulmonary manifestations are as deleterious as infectious pneumonia. A specific respiratory monitoring is necessary.

Immunologic assessment and KMT2D mutation detection in Kabuki syndrome.

Kabuki or Niikawa-Kuroki syndrome (KS) is a rare disorder with multiple malformations and recurrent infections, especially otitis media. This study aimed to investigate the genetic defects in Kabuki syndrome and determine if immune status is related to recurrent otitis media. Fourteen patients from 12 unrelated families were enrolled in the 9-year study period (2005-2013). All had Kabuki faces, cleft palate, developmental delay, mental retardation, and the short fifth finger. Recurrent otitis media (12/14) and hearing impairment (8/14) were also more common features. Immunologic analysis revealed lower memory CD19+ cells (11/13), lower memory CD4+ cells (8/13), undetectable anti-HBs antibodies (7/13), and antibody deficiency (7/13), including lower IgA (4), IgG (2), and IgG2 (1). Naïve emigrant lymphocytes, lymphocyte proliferation function, complement activity, and superoxide production in polymorphonuclear cells were all normal. All the patients had KMT2D mutations and 10 novel mutations of R1252X, R1757X,Y1998C, P2550R fs2604X, Q4013X, G5379X, E5425K, R5432X, R5432W, and R5500W. Resembling the phenotype of common variable immunodeficiency, KS patients with antibody deficiency, decreased memory cells, and poor vaccine response increased susceptibility to recurrent otitis media. Large-scale prospective studies are warranted to determine if regular immunoglobulin supplementation decreases the frequency of otitis media and severity of hearing impairment.

Design, synthesis and characterization of fMLF-mimicking AApeptides.

The tripeptide N-formyl-Met-Leu-Phe (fMLF) is a potent neutrophil chemoattractant and the reference agonist for the G protein-coupled N-formylpeptide receptor (FPR). As it plays a very important role in host defense and inflammation, there has been considerable interest in the development of fMLF analogues in the hope of identifying potential therapeutic agents. Herein we report the design, synthesis, and evaluation of AApeptides that mimic the structure and function of fMLF. The lead AApeptides induced calcium mobilization and mitogen-activated protein kinase (MAPK) signal transduction pathways in FPR-transfected rat basophilic leukemic (RBL) cells. More intriguingly, at high concentrations, certain AApeptides were more effective than fMLF in the induction of calcium mobilization. Their agonistic activity is further supported by their ability to stimulate chemotaxis and the production of superoxide in HL-60 cells. Similarly to fMLF, these AApeptides are much more selective towards FPR1 than FPR2. These results suggest that the fMLF-mimicking AApeptides might emerge as a new class of therapeutic agents that target FPRs.

Radiation promotes enucleation of erythroid cells possibly through the elevation of superoxide

The importance of Rac1 in the enucleation process of erythroid cells has previously been reported. We found that an inhibitor of Rac1 (NSC23766) could inhibit the enucleation of mouse erythroid progenitor cell line, MEDEP-BRC-5, which can differentiate into enucleated erythrocytes. It is known that Rac1 plays a central role in activating NADPH oxidase complex and NADPH oxidase produces superoxide in response to various signals and stresses. Among them, X-ray irradiation can induce production of superoxide in cells.

Alternative Splicing Generates a Diacylglycerol Kinase α Transcript That Acts as a Dominant‐Negative Modulator of Superoxide Production in Localized Aggressive Periodontitis

Diacylglycerol (DAG), levels of which are tightly regulated by diacylglycerol kinases (DGKs), is a lipid mediator linked to key biologic functions. Members of the DGK family undergo alternative splicing, generating the protein diversity necessary to control different intracellular DAG pools. DGKα function is altered in polymorphonuclear neutrophils (PMNs) of patients with localized aggressive periodontitis (LAgP), suggesting a genetic basis. Here, the authors assess DGKα spliced transcripts in human LAgP neutrophils. In an expression library of a patient with LAgP, PMNs were screened for different DGKα transcripts. Real-time polymerase chain reaction and in vitro expression assays were performed to assess the fate of different transcripts on protein translocation and superoxide production in human leukemia cells (HL-60) and COS-7 cells. A DGKα transcript that lacks exon 10 (DGKαΔ10) and generates a premature stop codon and a truncated protein was identified as being upregulated in LAgP neutrophils. In vitro assays revealed that DGKαΔ10 translocation occurred even in the absence of important regulatory motifs. Transfection of HL-60 neutrophil-like cells with the DGKαΔ10 spliced variant induced an increase in the stimulated production of superoxide anion replicating the phenotype of LAgP PMNs. DGKαΔ10 can act as a dominant-negative transcript that can modulate superoxide production and provides an example of genetic regulation of the inflammatory response that may be relevant to human inflammatory diseases such as LAgP.

넙치에서 분리된 phosphoinositide 3-kinase γ 유전자의 클로닝 및 특성 연구

Phosphoinositide 3-kinase (PI3K) plays a central role in cell signaling and leads to cell proliferation, survival, motility, exocytosis, and cytoskeletal rearrangements, as well as specialized cell responses, superoxide production, and cardiac myocyte growth. PI3K is divided into three classes; type I PI3K is preferentially expressed in leukocytes and activated by βγ subunits of heterotrimeric G-proteins. In this study, the cDNAs encoding the PI3Kγ gene were isolated from a brain cDNA library constructed using the flounder (Paralichthys olivaceus). The sequence of the isolated PI3Kγ was 1341 bp, encoding 447 amino acids. The nucleotide sequence of the PI3Kγ gene was analyzed with that of other species, including Oreochromis niloticus and Danio rerio, and it turned out to be well conserved during evolution. The PI3Kγ gene was subcloned into the expression vector pET-44a(+), and expressed in the E. coli BL21 (DE3) codon plus cell. The resulting protein was expressed as a fusion protein of approximately 49 kDa containing a C-terminal six-histidine extension that was derived from the expression vector. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to PI3Kγ. The binding of wortmannin to PI3Kγ, as detected by anti-wortmannin antisera, closely followed the inhibition of the kinase activities. The results obtained from this study will provide a wider base of knowledge on the primary structure and characterization of the PI3Kγ at the molecular level.

Pioglitazone reduces angiotensin II-induced COX-2 expression through inhibition of ROS production and ET-1 transcription in vascular cells from spontaneously hypertensive rats.

Glitazones have anti-inflammatory properties by interfering with the transcription of proinflammatory genes, such as cyclooxygenase (COX)-2, and with ROS production, which are increased in hypertension. This study analyzed whether pioglitazone modulates COX-2 expression in hypertension by interfering with ROS and endothelin (ET)-1. In vivo, pioglitazone (2.5 mg·kg(-1)·day(-1), 28 days) reduced the greater levels of COX-2, pre-pro-ET-1, and NADPH oxidase (NOX) expression and activity as well as O2 (·-) production found in aortas from spontaneously hypertensive rats (SHRs). ANG II increased COX-2 and pre-pro-ET-1 levels more in cultured vascular smooth muscle cells from hypertensive rats compared with normotensive rats. The ETA receptor antagonist BQ-123 reduced ANG II-induced COX-2 expression in SHR cells. ANG II also increased NOX-1 expression, NOX activity, and superoxide production in SHR cells; the selective NOX-1 inhibitor ML-171 and catalase reduced ANG II-induced COX-2 and ET-1 transcription. ANG II also increased c-Jun transcription and phospho-JNK1/2, phospho-c-Jun, and p65 NF-κB subunit nuclear protein expression. SP-600125 and lactacystin, JNK and NF-κB inhibitors, respectively, reduced ANG II-induced ET-1, COX-2, and NOX-1 levels and NOX activity. Pioglitazone reduced the effects of ANG II on NOX activity, NOX-1, pre-pro-ET-1, COX-2, and c-Jun mRNA levels, JNK activation, and nuclear phospho-c-Jun and p65 expression. In conclusion, ROS production and ET-1 are involved in ANG II-induced COX-2 expression in SHRs, explaining the greater COX-2 expression observed in this strain. Furthermore, pioglitazone inhibits ANG II-induced COX-2 expression likely by interfering with NF-κB and activator protein-1 proinflammatory pathways and downregulating ROS production and ET-1 transcription, thus contributing to the anti-inflammatory properties of glitazones.

Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Association of Phagocytic NADPH Oxidase Activity With Hypertensive Heart Disease

Left ventricular hypertrophy (LVH) is an independent marker of mortality in hypertension. Although the mechanisms contributing to LVH are complex, inflammation and oxidative stress may favor its development. We analyzed the association of the phagocytic NADPH oxidase-mediated superoxide anion release and LVH in patients with essential hypertension and the role of cardiotrophin-1 (CT-1) and interleukin-6 (IL-6), cytokines implicated in cardiac growth. Blood pressure, echocardiography data, and serum CT-1 and IL-6 levels were obtained in 140 subjects: 18 normotensives without LVH, 42 hypertensives without LVH, and 80 hypertensives with LVH. The NADPH oxidase-dependent superoxide production was assessed by chemiluminescence in peripheral blood mononuclear cells. Peripheral blood mononuclear cells were stimulated with CT-1 in vitro. Superoxide anion production by peripheral blood mononuclear cells associated with LVH and correlated with the left ventricular mass index. Serum CT-1 and IL-6 levels, which associated with the left ventricular mass index, correlated with superoxide production. Serum CT-1 and IL-6 levels were correlated. CT-1 stimulated NADPH oxidase superoxide production in peripheral blood mononuclear cells, which resulted in an increased release of IL-6. Our results show that superoxide anion production by the phagocytic NADPH oxidase associates with hypertensive heart disease, being significantly enhanced in hypertensive patients with LVH. This may be attributable to the activation of the NADPH oxidase by CT-1 and the subsequent release of IL-6. The phagocytic NADPH oxidase may be a therapeutic target in hypertensive heart disease.

Rac1 and Cholesterol Metabolism in Macrophage

In the early stages of atherosclerotic lesion development, cholesterol is mostly present as esterified cholesterol stored in macrophage cytoplasmic lipid droplets. However, when the lesion evolves, free cholesterol accumulates in other compartments, such as lysosomes and plasma membrane. A number of studies support a role for intracellular cholesterol content and distribution in regulating several cell functions. Particularly, membrane free cholesterol content has a specific effect on signaling pathways involved in regulating cell motility and organization of the actin cytoskeleton. These processes are regulated by several signaling pathways including the small GTPase Rac1. Rac1 belongs to the Rho GTPases of the Ras protein superfamily involved in the regulation of multiple cell functions, including cell proliferation, chemotaxis, phagocytosis, degranulation, and superoxide production. In this review, we discuss the role of Rac1 in macrophage with respect to cholesterol metabolism and trafficking as critical aspects for the development of atherosclerotic plaque.

Norepinephrine increases NADPH oxidase-derived superoxide in human peripheral blood mononuclear cells via α-adrenergic receptors

Many diseases associated with sympathoexcitation also exhibit elevated reactive oxygen species (ROS). A recent animal study indicated that exogenous administration of the sympathetic neurotransmitter norepinephrine (NE) increased systemic ROS via circulating leukocytes. The mechanisms contributing to this effect of NE and whether these findings can be translated to humans is unknown. Thus we tested the hypothesis that NE increases superoxide production in human peripheral blood mononuclear cells (PBMCs) via NADPH oxidase. Primary human PBMCs were freshly isolated from healthy young men and placed in culture. After NE (50 pg/ml, 50 ng/ml, and 50 μg/ml concentrations) or control treatments, NADPH oxidase mRNA expression (gp91(phox), p22(phox), and p67(phox)) was assessed using real-time RT-PCR, and intracellular superoxide production was measured using dihydroethidium fluorescence. PBMCs were also treated with selective adrenergic agonists-antagonists to determine the receptor population involved. In addition, CD14(+) monocyte-endothelial cell adhesion was determined using a fluorescent-based assay. NE significantly increased NADPH oxidase gene expression and intracellular superoxide production in a time-dependent manner (superoxide: 0.9 ± 0.2 fold, 6 h vs. 3.0 ± 0.3 fold, 36 h; NE, 50 μg/ml; P < 0.05). The sustained increase in NE-induced superoxide production was primarily mediated via α-adrenergic receptors, preferentially α2-receptors. The NADPH oxidase blocker diphenylene iodonium and protein kinase C inhibitor Staurosporine significantly attenuated NE-induced increases in superoxide production. Importantly, NE treatment increased CD14(+) monocyte-endothelial cell adhesion. These findings indicate for the first time that NE increases superoxide production in freshly isolated primary human PBMCs via NADPH oxidase through α-adrenergic receptors, an effect facilitating monocyte adhesion to the endothelium.

Activation of the mitochondrial ATP-sensitive K+ channel reduces apoptosis of spleen mononuclear cells induced by hyperlipidemia

BackgroundWe have previously demonstrated that increased rates of superoxide generation by extra-mitochondrial enzymes induce the activation of the mitochondrial ATP-sensitive potassium channel (mitoKATP) in the livers of hypertriglyceridemic (HTG) mice. The resulting mild uncoupling mediated by mitoKATP protects mitochondria against oxidative damage. In this study, we investigate whether immune cells from HTG mice also present increased mitoKATP activity and evaluate the influence of this trait on cell redox state and viability.MethodsOxygen consumption (Clark-type electrode), reactive oxygen species production (dihydroethidium and H2-DCF-DA probes) and cell death (annexin V, cytocrome c release and Trypan blue exclusion) were determined in spleen mononuclear cells.ResultsHTG mice mononuclear cells displayed increased mitoKATP activity, as evidenced by higher resting respiration rates that were sensitive to mitoKATP antagonists. Whole cell superoxide production and apoptosis rates were increased in HTG cells. Inhibition of mitoKATP further increased the production of reactive oxygen species and apoptosis in these cells. Incubation with HTG serum induced apoptosis more strongly in WT cells than in HTG mononuclear cells. Cytochrome c release into the cytosol and caspase 8 activity were both increased in HTG cells, indicating that cell death signaling starts upstream of the mitochondria but does involve this organelle. Accordingly, a reduced number of blood circulating lymphocytes was found in HTG mice.ConclusionsThese results demonstrate that spleen mononuclear cells from hyperlipidemic mice have more active mitoKATP channels, which downregulate mitochondrial superoxide generation. The increased apoptosis rate observed in these cells is exacerbated by closing the mitoKATP channels. Thus, mitoKATP opening acts as a protective mechanism that reduces cell death induced by hyperlipidemia.

Involvement of small GTPase RhoA in the regulation of superoxide production in BV2 cells in response to fibrillar Aβ peptides

Fibrillar amyloid-beta (fAβ) peptide causes neuronal cell death, which is known as Alzheimer's disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAβ. We observed that fAβ rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAβ1–42 and fAβ25–35 induced superoxide production in BV2 cells. fAβ25–35 produced superoxide, although fAβ25–35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAβ does not seem to be dependent on phagocytosis of fAβ. Herein we studied how fAβ produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAβ. Furthermore, fAβ elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47PHOX. Moreover, peritoneal macrophages from p47PHOX (−/−) knockout mouse could not produce superoxide in response to fAβ. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAβ through phosphorylation of p47PHOX in microglial BV2 cells.

Resveratrol prevents hypertension and cardiac hypertrophy in hypertensive rats and mice

Resveratrol (RESV) is a polyphenol with pleiotropic effects that include reduction of oxidative stress and increased vascular nitric oxide (NO) production. However, whether or not RESV can prevent rises in blood pressure (BP) is controversial and remains to be firmly established. The purpose of this study was to determine whether RESV attenuates elevated BP and subsequent adaptive cardiac hypertrophy and to better understand the mechanisms involved. The spontaneously hypertensive rat (SHR) and the angiotensin (Ang)-II infused mouse were used as hypertensive models. Compared to a standard control diet, consumption of diets containing RESV by SHRs and Ang-II hypertensive mice, markedly prevented rises in systolic BP. In addition, flow-mediated vasodilation was significantly improved by RESV in SHRs. RESV also reduced serum and cardiac levels of the lipid peroxidation by-product, 4-hydroxy-2-nonenal in the hypertensive rodents and inhibited the production of superoxide in human-derived endothelial cells. Analysis of mesenteric arteries from SHRs and Ang-II infused mice demonstrated that RESV increased endothelial NO synthase (eNOS) phosphorylation by enhancing the LKB1/adenosine monophosphate (AMP)-activated protein kinase (AMPK) signal transduction pathway. Moreover, RESV reduced hypertrophic growth of the myocardium through reduced hemodynamic load and inhibition of the p70 S6 kinase pro-hypertrophic signaling cascade. Overall, we show that high dose RESV reduces oxidative stress, improves vascular function, attenuates high BP and prevents cardiac hypertrophy through the preservation of the LKB1–AMPK–eNOS signaling axis.

Modulation of ceramide-induced cell death and superoxide production by mitochondrial DNA-encoded respiratory chain defects in Rattus xenocybrid mouse cells

Mitochondria play an integral role in cell death signaling, yet how mitochondrial defects disrupt this important function is not well understood. We have used a mouse L-cell fibroblast model harboring Rattus norvegicus mtDNA (Rn xenocybrids) to examine the effects of multiple oxidative phosphorylation (OXPHOS) defects on reactive oxygen species (ROS) generation and cell death signaling. Blue native-PAGE analyses of Rn xenocybrids revealed defects in OXPHOS complex biogenesis with reduced steady-state levels of complexes I, III and IV. Isolated Rn xenocybrid mitochondria exhibited deficiencies in complex II+III and III activities, with CIII-stimulated ROS generation 66% higher than in control mitochondria. Rn xenocybrid cells were resistant to staurosporine-induced cell death, but exhibited a four-fold increase in sensitivity to ceramide-induced cell death that was caspase-3 independent and did not induce chromosomal DNA degradation. Furthermore, ceramide directly inhibited Rn xenocybrid complex II+III activity by 97%, although this inhibition could be completely abolished by exogenous decylubiquinone. Ceramide also induced a further increase in ROS output from Rn xenocybrid complex III by 42%. These results suggest that the interaction of ceramide with OXPHOS complex III is significantly enhanced by the presence of the xenotypic Rattus cytochrome b in complex III, likely due to the increased affinity for ceramide at the ubiquinone binding site. We propose a novel mechanism of altered mitochondrial cell death signaling due to mtDNA mutations whereby ceramide directly induces OXPHOS complex ROS generation to initiate cell death pathways.

Effects of Aging and Hypercholesterolemia on Oxidative Stress and DNA Damage in Bone Marrow Mononuclear Cells in Apolipoprotein E-deficient Mice

Recent evidence from apolipoprotein E-deficient (apoE−/−) mice shows that aging and atherosclerosis are closely associated with increased oxidative stress and DNA damage in some cells and tissues. However, bone marrow cells, which are physiologically involved in tissue repair have not yet been investigated. In the present study, we evaluated the influence of aging and hypercholesterolemia on oxidative stress, DNA damage and apoptosis in bone marrow cells from young and aged apoE−/− mice compared with age-matched wild-type C57BL/6 (C57) mice, using the comet assay and flow cytometry. The production of both superoxide and hydrogen peroxide in bone marrow cells was higher in young apoE−/− mice than in age-matched C57 mice, and reactive oxygen species were increased in aged C57 and apoE−/− mice. Similar results were observed when we analyzed the DNA damage and apoptosis. Our data showed that both aging and hypercholesterolemia induce the increased production of oxidative stress and consequently DNA damage and apoptosis in bone marrow cells. This study is the first to demonstrate a functionality decrease of the bone marrow, which is a fundamental extra-arterial source of the cells involved in vascular injury repair.

Crumbs limits oxidase-dependent signaling to maintain epithelial integrity and prevent photoreceptor cell death

Drosophila melanogaster Crumbs (Crb) and its mammalian orthologues (CRB1-3) share evolutionarily conserved but poorly defined roles in regulating epithelial polarity and, in photoreceptor cells, morphogenesis and stability. Elucidating the molecular mechanisms of Crb function is vital, as mutations in the human CRB1 gene cause retinal dystrophies. Here, we report that Crb restricts Rac1-NADPH oxidase-dependent superoxide production in epithelia and photoreceptor cells. Reduction of superoxide levels rescued epithelial defects in crb mutant embryos, demonstrating that limitation of superoxide production is a crucial function of Crb and that NADPH oxidase and superoxide contribute to the molecular network regulating epithelial tissue organization. We further show that reduction of Rac1 or NADPH oxidase activity or quenching of reactive oxygen species prevented degeneration of Crb-deficient retinas. Thus, Crb fulfills a protective role during light exposure by limiting oxidative damage resulting from Rac1-NADPH oxidase complex activity. Collectively, our results elucidate an important mechanism by which Crb functions in epithelial organization and the prevention of retinal degeneration.

Abstract 368: Dynamic Receptor-Ligand Activation of Nox1 Regulates Blood Flow via a Novel TSP1-CD47 Signaling Axis

Reactive oxygen species (ROS) play an important role in ischemia-reperfusion injury but the mechanisms controlling this phenomenon remain elusive. Our previous studies demonstrated that matrix protein thrombospondin-1 (TSP1) at concentrations found in human disease (2.2 nM) is a rapid and potent stimulator of superoxide (O2•–) production in vascular smooth muscle cells (VSMC). However, the mechanism by which TSP1 mediates this response and its role in reperfusion-induced impairment of tissue perfusion are unknown. Here, we show that blockade of integrin-associated CD47 signaling (pmol O2•–/mg protein/min: 6.7±0.6, 10.2±0.7*, 6.0±0.5, and 7.1±0.7 for isotype control + vehicle, isotype control + TSP1, CD47 blocking antibody + vehicle and CD47 blocking antibody + TSP1; *p&lt;0.05 vs isotype control + vehicle) and gene silencing of NADPH oxidase 1 (Nox1) abrogated TSP1-stimulated O2•– in VSMC. By luciferase reporter array, we identified myocyte enhancer factor 2 (MEF2) as a downstream effector of TSP1 and show that knockdown of MEF2A suppresses TSP1-stimulated O2•– (pmol O2•–/mg protein/min: 4.2±0.3, 6.3±0.4*, 4.1±0.3, and 4.6±0.3 for scrambled siRNA + vehicle, scrambled siRNA + TSP1, MEF2A siRNA + vehicle and MEF2A siRNA + TSP1;*p&lt;0.05 vs scrambled siRNA + vehicle). Further, TSP1 rapidly phosphorylated the Nox organizer subunit p47phox in VSMC. Electron paramagnetic resonance demonstrated that TSP1 stimulates O2•– in wild type (WT), but not in Nox1y/-, endothelium-free aortic rings (CM• radical formation (x10 3): 20.9±11.1, 86.8±20.5*, 20.2±28.6 and 29.6±22.2 for WT + vehicle, WT + TSP1, Nox1y/- + vehicle, and Nox1y/- + TSP1; *p&lt;0.05 vs WT + vehicle). Finally, intravenous TSP1 acutely decreased recovery of hindlimb blood flow following ischemia-reperfusion as measured by real time laser Doppler in control, but not in Nox1 gene silenced, rats. Our findings suggest a highly-regulated role of matricellular TSP1 as a stimulator of a novel CD47-MEF2A-Nox1 signaling axis and ROS and support a role for the matrix protein as a blood flow attenuator in cardiovascular disease.