Role Of NAD Research Articles

Abstract 570: Role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in the cytotoxicity of the Hsp90 inhibitor 17AAG in human pancreatic cancer cells

Abstract Previous work has demonstrated that NQO1 can reduce the benzoquinone ansamycin Hsp90 inhibitor 17-allyamino-demethoxy geldanamycin (17AAG) to the corresponding hydroquinone (17AAGH2). The formation of 17AAGH2 by NQO1 results in a more water soluble molecule and that binds with greater affinity to Hsp90 when compared to the parent quinone 17AAG. Experiments were conducted with the human pancreatic cancer cell line Panc1 which is homozygous for the NQO1*2 polymorphism and contains only trace levels of NQO1 protein and no NQO1 catalytic activity. An isogenic cell line expressing high levels of NQO1 (Panc1-C5) was generated by stable transfection of wild type NQO1 in Panc1 cells. 17AAG was more cytotoxic to Panc1-C5 cells with high levels of NQO1 when compared to NQO1-null parental Panc1 cells. Pretreatment of Panc1-C5 cells with the NQO1 mechanism-based inhibitor ES936 reduced the cytotoxicity of 17AAG to the level observed in Panc1 cells. No difference in the cytotoxicity of the non-quinone Hsp90 inhibitor radicicol was observed between the two cell lines Biomarkers of Hsp90 inhibition were measured in both Panc1 and Panc1-C5 cells following treatment with 17AAG. Higher protein levels of Hsp70 and lower amounts of cRaf, phosphorylated ERK1/2 and phosphylated MEK were observed in Panc1-C5 cells compared to Panc1 cells. In addition, cell cycle analysis revealed a greater number of Panc1-C5 cells arrested in G2M following 17AAG treatment compared to Panc1 cells. Cytotoxicity studies were also performed in additional human pancreatic cancer cell lines (BxPC3 and MiaPaCa2) that contain high levels of catalytically active NQO1*1 protein and in agreement with the studies described above the cytotoxicity of 17AAG could be reduced significantly following pretreatment with the NQO1 inhibitor ES936. The intracellular concentrations of 17AAG and 17AAH2 were measured in human pancreatic cancer cell lines and it was observed that greater amounts of both 17AAG and 17AAGH2 could be detected in cell lines with catalytically active NQO1 when compared to cells lacking NQO1 activity or cells pretreated with ES936. These data demonstrate that in addition to generating a more potent Hsp90 inhibitor (17AAGH2) the reduction of 17AAG to 17AAGH2 by NQO1 leads to significantly greater intracellular concentrations of 17AAG and 17AAGH2. These data confirm the role of NQO1 in sensitivity to 17AAG and suggest that genotyping for the NQO1*2 polymorphism should be considered in patients undergoing 17AAG treatment for pancreatic cancer or other solid tumors that are known to contain high levels of NQO1 activity (Supported by CA51210). 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 570.

Abstract 402: Role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in chemotherapy induced myeloid leukemia

Abstract Individuals with NQO1P187S mutation have no detectable NQO1 protein levels. These individuals have been extensively predicted to be at high risk for developing chemotherapy-induced leukemia. We have studied the effect of Etoposide, a DNA Topoisomerase II inhibitor used in many chemotherapeutic regimens, on NQO1 gene deleted (NQO1-/-) mice. NQO1-/- mice are born with a myelodysplastic bone marrow. Flow cytometric analysis of blood, bone marrow and spleen showed an increase in megakaryocytic and myeloid cell lineages in NQO1-/- mice. A spectrum of responses ranging from leukopenia to myeloid leukemia were observed with increasing drug concentration by Flow cytometry and confirmed by histochemistry in NQO1-/- mice. C/EBPalpha plays a key role in the myeloid lineage commitment of hematopoietic progenitor cells. NQO1 protects C/EBPalpha from 20S proteasomal degradation in in vitro assays. Altered C/EBPalpha levels are responsible for the preferential increase in myeloid cell lineage in NQO1-/- mice. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 402.

Abstract 68: Nampt regulates fatty acid synthesis, ER homeostasis, and survival in prostate cancer

Abstract Nicotinamide adenine dinucleotide (NAD) is crucial for the survival of living organisms. This coenzyme functions in both redox metabolism and as a substrate for several proteins, notably Sirtuin deacetylases and poly-(ADP ribose) polymerases, which control stress response and apoptosis in cancer. The rate limiting step of NAD biosynthesis is catalyzed by Nicotinamide phosphoribosyl transferase (Nampt). Recent evidence suggests Nampt may play a role in cancer by regulating survival. The Nampt inhibitor FK866 is in phase II clinical trials against a variety of solid malignancies, however no studies have focused on the role of NAD in prostate cancer (PCa). Because tumor cells have a high requirement for NAD and PCa has increased fatty acid synthesis, we hypothesized that Nampt is important for PCa cell survival and metabolism. Our studies demonstrate that Nampt activity is required for the survival of prostate tumor cells (PC-3, LNCaP, and DU145), but not normal prostate epithelial cells, as determined by treatment with FK866. Exogenous NAD rescued cell death, indicating FK866 is a specific inhibitor of Nampt. To test whether PCa cells can rely on de novo NAD synthesis, we treated cell lines with FK866 and added back nicotinic acid, the substrate for the de novo pathway. Only DU145 cell survival was rescued by nicotinic acid demonstrating that NAD biosynthesis pathways are heterogeneous in PCa. Furthermore, expression of Nicotinic acid phosphoribosyl transferase (Naprt1), the rate-limiting step of the de novo pathway, correlated with the rescue by nicotinic acid in DU145 cells. Additionally, Nampt inhibition activated endoplasmic reticulum (ER) stress and autophagy in a pro-survival manner in all cell lines. Interestingly, FK866 also dramatically reduced fatty acid synthesis in PC-3 and DU145 cells, independent of fatty acid synthase levels. However, LNCaP cells showed no decrease in 14C-acetate incorporation. Robust phosphorylation of ACC occured in DU145 and PC-3 cells upon treatment with FK866, and this effect was rescued by adding back NAD. This correlated with reduced ATP levels and activation (phosphorylation) of AMPK at T172. LNCaP cells had a less dramatic depletion of ATP and no changes in AMPK or ACC phosphorylation, indicating a potential mechanism of fatty acid synthesis shutdown. However, lipid synthesis (as measured by incorporation of 14C-choline) was reduced in all 3 cell lines, albeit to a lesser extent in LNCaP cells. Overall, these findings demonstrate a novel role for Nampt in PCa, and highlight the contribution of NAD to ER homeostasis, fatty acid synthesis, and survival. Targeting NAD biosynthesis could be a novel approach to disrupting PCa cell survival and lipid synthesis. 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 68.

Review: NAD + : A modulator of immune functions

Latterly, nicotinamide adenine dinucleotide (NAD+) has emerged as a molecule with versatile functions and of enormous impact on the maintenance of cell integrity. Besides playing key roles in almost all major aspects of energy metabolism, there is mounting evidence that NAD+ and its degradation products affect various biological activities including calcium homeostasis, gene transcription, DNA repair, and intercellular communication. This review is aimed at giving a brief insight into the life cycle of NAD+ in the cell, referring to synthesis, action and degradation aspects. With respect to their immunological relevance, the importance and function of the major NAD+ metabolizing enzymes, namely CD38/CD157, ADP-ribosyltransferases (ARTs), poly-ADP-ribose-polymerases (PARPs), and sirtuins are summarized and roles of NAD+ and its main degradation product adenosine 5'-diphosphoribose (ADPR) in cell signaling are discussed. In addition, an outline of the variety of immunological processes depending on the activity of nicotinamide phosphoribosyltransferase (Nampt), the key enzyme of the salvage pathway of NAD+ synthesis, is presented. Taken together, an efficient supply of NAD+ seems to be a crucial need for a multitude of cell functions, underlining the yet only partly revealed potency of this small molecule to influence cell fate.

Correction: Mach, J., et al. The Effect of Antioxidant Supplementation on Fatigue during Exercise: Potential Role for NAD+(H). Nutrients 2010, 2, 319-329

We have found an error in our manuscript published in Nutrients [...]

NAD+ can induce autophagy of Neuro2a cells and PC12 cells

Autophagy is an important form of programmed cell death which could play significant roles in the pathology of multiple diseases. NAD+ is a fundamental molecule in various biological functions. However, it is unknown what is the role of NAD+ in autophagy. The goal of this study is to determine the effects of NAD+ on autophagy using Neuro2a cells and PC12 cells as cell models. We assessed the levels of autophagy by conducting MDC staining assay, real time PCR assay of beclin‐1, and Western blot of LC 3. We found that treatment of 1–10 mM NAD+ produced significant increases in MDC staining and beclin‐1 mRNA levels of Neuro2a cells, as well as significant increases in MDC staining and the ratios of LC 3 II/LC 3 I in PC 12 cells. These studies suggest that NAD+ treatment can induce autophagy of these types of cells. We further found that nicotinamide ‐‐‐ the degradation product of NAD+‐‐‐ could not affect the levels of autophagy, thus arguing against the possibility that NAD+ induces autophagy through generating nicotinamide. In summary, our study indicates that NAD+ can induce autophagy of such cell types as Neuro2a cells and PC12 cells, suggesting a new pathway of inducing autophagy as well as a novel property of NAD+ (Supported by the Key Research Grant of Shanghai Municipal Education Committee, Pujiang Scholar Program Award and a 973 Grant (to W.Y.)).

Time‐course effects of low salt diet on the regulation of cardiac NO bioactivity: Role of NAD(P)H oxidase

The time‐course effects of low salt diet (LSD) on ANG II, MVO2 and NO bioactivity were studied in gp91phox (−/−) (KO) mice. A 2 week LSD significantly (P<0.05) increased the plasma ANG II levels in both wild type (WT) and KO mice. An 8 week LSD also increased plasma ANG II levels in WT (355±77 to 508±36pg/ml, P<0.05, n=10) but there was no change in KO mice. On normal diet (ND), stimulation of NO production by bradykinin (BK) induced a concentration dependent reduction in MVO2 (−32±2% and −25±2%) in vitro in both WT and KO mice respectively. The reduction in MVO2 was significantly (P<0.0001) attenuated by L‐NAME in WT and KO mice. BK‐induced reduction in MVO2 was significantly attenuated in WT (−14±1%, P<0.0001) on 8 week LSD but not in KO mice. The BK‐induced reduction in MVO2 was restored in WT by apocynin (−25±1%), by losartan (−25±1%) or by tiron (−28±2%). Similar effects were seen in KO mice on 2 week LSD. Systolic and diastolic blood pressure (BP) was significantly increased after 8 week LSD in WT (123±2/79±1 to 143±4/90±3, P<0.01, n=10). No significant increase in BP was seen in KO mice on 8 week LSD or WT and KO mice on 2 week LSD. Protein expressions of gp91 and p47 increased significantly (P<0.05) in WT on 8 week LSD. Thus LSD increases plasma ANG II and reduces NO bioactivity in the heart. The assembly and activation of NAD(P)H oxidase by ANG II may be the basis of LSD induced cardiovascular dysfunction. Supported by PO‐1 HL 43023 and 50142.

Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.

Dealcoholized red wine reverse vascular remodeling in an experimental model of metabolic syndrome: role of NAD(P)H oxidase and eNOS activity

The present study examines the effect of chronic administration of dealcoholized red wine Malbec (DRW) on vascular remodeling and NAD(P)H oxidase and endothelial nitric oxide synthase activity (eNOS) in an experimental model of metabolic syndrome induced by fructose administration. Thirty-day old male Wistar rats were fed a normal rat diet (control) or the same diet plus 10% fructose in drinking water (FFR). During the last 4 weeks of a 10-week period of the corresponding diet, a subgroup of control and FFR (n=8 each) received DRW in their drinking water. Systolic blood pressure (SBP), a homeostasis model assessment of insulin resistance (HOMA-IR), aortic NAD(P)H oxidase and eNOS activity in the heart and vascular tissue were evaluated. Vascular remodeling was evaluated in the left carotid artery (CA) and interlobar, arcuate and interlobular renal arteries (RA) through lumen to media (L/M) ratio determination. At the end of the study FFR increased the SBP (p < 0.001), HOMA-IR (p < 0.001), and aortic NAD(P)H oxidase activity (p < 0,05) but reduced cardiac and vascular eNOS activity (p < 0.01), L/M ratio in CA (p < 0.001) and RA (p < 0.01) compared with the C group. DRW reduced SBP (p < 0.05), aortic NAD(P)H oxidase (p < 0.05), and recovered eNOS activity (p < 0.001) and L/M in CA (p < 0.001) and RA (p < 0.001) compared with FFR. This study provides new data about the beneficial effect of DRW on oxidative stress and vascular remodeling in the experimental model of metabolic syndrome. Data suggest the participation of mechanisms involving oxidative stress in FFR alterations and the usefulness of natural antioxidant substances present in red wine in the reversion of these changes.

Mechanistic studies on the effects of nicotinamide on megakaryocytic polyploidization and the roles of NAD + levels and SIRT inhibition

Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood CD34(+) cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy. We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mobilized peripheral blood CD34(+) cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD(+) precursors. We tested additional inhibitors of the sirtuin (or SIRT) 1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets. NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure; 6.25 mM NIC increased NAD(+) levels fivefold. Quinolinic acid increased NAD(+) similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. NIC greatly increases Mk ploidy under a wide range of conditions without altering Mk morphology. Inhibition of SIRT1 and/or SIRT2 is primarily responsible for NIC effects on Mk maturation.

NAD(P)H Oxidase-Dependent Regulation of CCL2 Production during Retinal Inflammation

CCL2 plays an important role in vascular inflammation by inducing leukocyte recruitment and activation. The authors had previously found that the blockade of NAD(P)H oxidase in turn blocks leukocyte adhesion to retinal vessels during diabetes and uveitis. In this study, the role of NAD(P)H oxidase in CCL2 production was assessed. Studies were performed in three mouse models with lipopolysaccharide (LPS)-induced uveitis, ischemic retinopathy, and streptozotocin diabetes and in cytokine- and LPS-treated cells. CCL2 mRNA and protein expression were measured by quantitative PCR and ELISA. NF-kappaB activity was detected by reporter gene assay. Kinase phosphorylation was determined by immunoblotting. Expression of CCL2 was increased in the retinas of all three mouse models. The effect was strongest in the LPS-treated mice, with a peak mRNA increase at 3 hours. This increase was abrogated by administration of the NAD(P)H oxidase inhibitor apocynin. Apocynin also blocked CCL2 production in endothelial cells (ECs), retinal microglia, and Müller cells stimulated with TNF-alpha, VEGF, or LPS. Studies using human ECs demonstrated that TNF-alpha-induced CCL2 production was also inhibited by the NAD(P)H oxidase inhibitor DPI, the antioxidant N-acetyl-L-cysteine, or the superoxide scavenger Tiron, further indicating that inhibition occurs through the NAD(P)H/ROS pathway. Analysis of downstream signals showed that inhibition of NAD(P)H oxidase partially inhibited NF-kappaB activation but did not reduce CCL2 mRNA stability or prevent TNF-alpha-induced phosphorylation of p38MAPK. However, TNF-alpha-induced Akt phosphorylation was blocked, and inhibiting Akt dramatically decreased CCL2 production. NAD(P)H oxidase activity is required for CCL2 production during retinal vascular inflammation. Akt and NF-kappaB are involved in this signaling pathway.

Increased cerebral ischemic damage during chronic alcohol consumption: role of NAD(P)H oxidase

We hypothesized that NAD(P)H oxidase may contribute to increased cerebral ischemic damage during chronic alcohol consumption. Sprague‐Dawley rats were randomly divided into four groups: nonalcohol‐fed, alcohol‐fed, nonalcohol‐fed plus apocynin‐treated, and alcohol‐fed plus apocynin‐treated. Alcohol (8 g/kg/day) was mixed into a liquid diet and given to rats for 8 weeks. Apocynin (7.5 mg/kg/day) was orally administrated for 4 weeks prior to the experiment. Rats were subjected to 2 hours of right middle cerebral artery occlusion (MCAO). Infarct volume and neurological score were assessed 24 hours after reperfusion. Superoxide production from ischemic border area was measured by lucigenin‐enhanced chemiluminescence. In addition, expression of NAD(P)H oxidase membrane subunit gp91phox in ischemic border area was analyzed by Western blot. Compared with nonalcohol‐fed rats, alcohol‐fed rats had a larger infarct volume, worse neurological score and higher superoxide production. Apocynin did not alter infarct size, neurological deficit or superoxide production in nonalcohol‐fed rats, but significantly reduced infarct size, improved neurological outcome and attenuated superoxide production in alcohol‐fed rats. Chronic alcohol consumption significantly increased gp91 phox expression compared with nonalcohol‐fed rats, and cerebral ischemia also significantly enhanced gp91 phox expression compared with nonischemic side. Apocynin treatment did not alter gp91 phox expression in either nonalcohol‐fed rats or alcohol‐fed rats. Our findings suggest that NAD(P)H oxidase may play an important role in increased cerebral ischemic damage during chronic alcohol consumption.

Role of oxidative stress in the natriuresis induced by central administration of angiotensin II

Central administration of angiotensin II (Ang II) is known to reduce urinary volume and to increase sodium and potassium excretion. Recently, a novel signalling mechanism for Ang II in the periphery has been shown to involve reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase-derived reactive oxygen species (ROS).Although ROS are now known to be involved in numerous Ang II-regulated processes in peripheral tissues, and are increasingly implicated in CNS neurodegenerative diseases, the role of ROS in central regulation of Ang II-induced hydromineral metabolism remains unexplored.The hypothesis that ROS are involved in central Ang II signalling and in Ang II-dependent antidiuresis, natriuresis and kaliuresis was tested by the use of selective antagonists of the NAD(P)H oxidase cascade. In intracerebroventricular (ICV)-cannulated rats,Ang II was injected ICV and urinary sodium and potassium excretion was assessed at 1-, 3-, and 6-hour periods of urine collection. Urine sample was analysed for sodium and potassium concentration using a flame photometer. The role of NAD(P)H oxidase-dependent signalling cascade was evaluated using the selective NAD(P)H oxidase inhibitor, apocynin; the superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol); and the protein kinase C inhibitor, chelerythrine. ICV administration of Ang II to conscious hydrated rats resulted in a significant decrease in urinary volume in the first hour, and an increased sodium and potassium excretion during the 6-hour period of urine collection, which was most effective during the 3 and 6 h. Interference with the NAD(P)H oxidase signalling by central administration of apocynin, tempol or chelerythrine, blunted the natriuretic and kaliuretic effect induced by central administration of Ang II, without affecting its antidiuretic action. This study demonstrates that increases of urinary sodium and potassium excretion elicited by central administration of Ang II are mediated by NAD(P)H oxidase- dependent production of superoxide and protein kinase C activation in conscious hydrated rats.

Roles of NAD / NADH and NADP+ / NADPH in Cell Death

A rapidly growing body of information has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could be new fundamental factors in cell death: Many studies have indicated key roles of poly (ADP-ribose) polymerases and sirtuins--two families of NAD-dependent enzymes--in cell death; and NAD may also affect cell survival by influencing mitochondrial permeability transition, apoptosis-inducing factor and GAPDH. NAD may further influence cell survival by its effects on calcium homeostasis, gene expression and immunological functions. Due to the crucial roles of oxidative stress in cell death, NADPH may mediate cell death by its major effects on oxidative stress: NADPH is a key factor in cellular antioxidation systems; and NADPH oxidase is also a major generator of oxidative stress. With growing information about the novel biological properties of NAD and NADP, it is likely that new roles of NAD and NADP in cell death and various diseases will be elucidated. The elucidation may not only improve our understanding about the fundamental mechanisms of cell death, but also suggest new therapeutic targets for a variety of diseases.

Cellular Senescence: Molecular Mechanisms,In VivoSignificance, and Redox Considerations

Cellular senescence is recognized as a critical cellular response to prolonged rounds of replication and environmental stresses. Its defining characteristics are arrested cell-cycle progression and the development of aberrant gene expression with proinflammatory behavior. Whereas the mechanistic events associated with senescence are generally well understood at the molecular level, the impact of senescence in vivo remains to be fully determined. In addition to the role of senescence as an antitumor mechanism, this review examines cellular senescence as a factor in organismal aging and age-related diseases, with particular emphasis on aberrant gene expression and abnormal paracrine signaling. Senescence as an emerging factor in tissue remodeling, wound repair, and infection is considered. In addition, the role of oxidative stress as a major mediator of senescence and the role of NAD(P)H oxidases and changes to intracellular GSH/GSSG status are reviewed. Recent findings indicate that senescence and the behavior of senescent cells are amenable to therapeutic intervention. As the in vivo significance of senescence becomes clearer, the challenge will be to modulate the adverse effects of senescence without increasing the risks of other diseases, such as cancer. The uncoupled relation between cell-cycle arrest and the senescent phenotype suggests that this is an achievable outcome.

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Choroidal neovascularization (CNV) occurs in a variety of chorioretinal diseases including age-related macular degeneration (AMD), and is the major cause of severe visual loss in patients with AMD. Oxidative stress has been thought to play an important role in the development of CNV. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of the major intracellular sources of reactive oxygen species (ROS) in the vascular system. In this study, we examined the expression of p22phox, an integral subunit in the NADPH oxidase complex, in the mouse eye. We determined that p22phox is expressed in the retinal pigment epithelial (RPE) cells and inner retinal neurons. A small-interfering RNA (siRNA) designed against p22phox efficiently reduced the expression of the protein in the eye when delivered by means of recombinant adeno-associated virus (AAV) vector. Vector treatment inhibited CNV in the mouse when delivered into the subretinal space where RPE cells were transduced. These results suggest that NADPH oxidase-mediated ROS production in RPE cells may play an important role in the pathogenesis of neovascular AMD, and that this pathway may represent a new target for therapeutic intervention in AMD.

Role of NAD(P)H Oxidase‐ and Mitochondria‐derived ROS in Coronary Collateral Growth

Relative importance of NAD(P)H oxidase‐ vs. mitochondria (mt)‐derived oxidative stress (OxStress) in cardiovascular pathologies, including impaired arteriogenesis, is unresolved. We investigated the contribution both to regulation of coronary collateral growth (CCG). CCG was stimulated in lean Zucker (LZR) and prediabetic obese Zucker (ZOF), where OxStress is elevated, rats by repetitive ischemia (RI). Coronary blood flow was measured in normal (NZ) and collateral‐dependent (CZ) zones. RI increased CZ flow (0.23 to 0.84 ml/min/g; CZ/NZ 0.84 ± 0.03) in LZR, but failed to increase it in ZOF (0.08 to 0.15 ml/min/g; CZ/NZ 0.16 ± 0.03). Treatment with a ubiquitous ROS scavenger, TEMPOL, partially restored CCG in ZOF (CZ/NZ 0.69 ± 0.05). Treatment with mt‐targeted TEMPOL and another mt‐targeted ROS scavenger (Mito‐Quinone) restored RI‐induced CCG in ZOF to a similar extent (CZ/NZ 0.64 ± 0.01 and 0.75 ± 0.04, respectively). Treatment with the NAD(P)H oxidase inhibitor, apocynin also partially rescued CCG in ZOF and in a rat model of the metabolic syndrome (JCR), where OxStress is similarly elevated and CCG compromised (CZ/NZ 0.50 ± 0.02 and 0.65 ± 0.03, respectively). We conclude that both NAD(P)H oxidase‐ and mt‐derived OxStress are important in the regulation of CCG, and lowering of both may be necessary for restoration of CCG in the prediabetic metabolic syndrome. Support: AHA 0630285N, NIH RR018766, NIH 32788.

Human Immunodeficiency Virus Type 1 Tat Protein Inhibits the SIRT1 Deacetylase and Induces T Cell Hyperactivation

Symptoms of T cell hyperactivation shape the course and outcome of HIV-1 infection, but the mechanism(s) underlying this chronic immune activation are not well understood. We find that the viral transactivator Tat promotes hyperactivation of T cells by blocking the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1. Tat directly interacts with the deacetylase domain of SIRT1 and blocks the ability of SIRT1 to deacetylate lysine 310 in the p65 subunit of NF-kappaB. Because acetylated p65 is more active as a transcription factor, Tat hyperactivates the expression of NF-kappaB-responsive genes, a function lost in SIRT1-/- cells. These results support a model where the normal function of SIRT1 as a negative regulator of T cell activation is suppressed by Tat during HIV infection. These events likely contribute to the state of immune cell hyperactivation found in HIV-infected individuals.

NAD+ and NADH in ischemic brain injury

NAD+ and NADH have been emerging as the common mediators of energy metabolism, mitochondrial functions, calcium homeostasis, aging and cell death. NAD+ and NADH can affect cell death by various mechanisms, such as influencing energy metabolism, mitochondrial permeability transition pores, and apoptosis-inducing factor. Because energy failure, calcium disregulation and cell death are the key components in the tissue damaging cascade initiated by cerebral ischemia, it is likely that NAD+ and NADH play significant roles in ischemic brain damage. Many studies, including the findings that poly(ADP-ribose) polymerase-1 mediates ischemic brain injury and that NAD+ administration can decrease ischemic brain damage, have suggested significant roles of NAD+ and NADH in the debilitating illness. However, there is still distinct insufficiency of the information regarding the roles of NAD+ and NADH in ischemic brain injury. Because increasing evidence has indicated critical functions of NAD+ and NADH in various biological processes, future studies on the roles of NAD+ and NADH in cerebral ischemia may expose essential mechanisms underlying ischemic brain injury and suggest novel therapeutic strategies for the illness.

A role of NAD in life-span extension control

A role of NAD in life-span extension control