1、Acta Physiologica Sinica, April 25, 2007, 59 (2): 117-127117Brief ReviewNrf2/ARE regulated antioxidant gene expression in endothelial and smoothmuscle cells in oxidative stress: implications for atherosclerosis andpreeclampsiaGiovanni E. Mann1,*, Jörg Niehueser-Saran1, Alan Watson1, Ling Gao1,

2、Tetsuro Ishii2, Patricia de Winter1, RichardC. M. Siow12Cardiovascular Division, School of Medicine, Kings College London, 150 Stamford Street, London SE1 9NH, United Kingdom;Department of Molecular and Cellular Physiology, Graduate School of Comprehensive Human Sciences, University of Tsukuba,Ibara

3、ki 305-8577, JapanAbstract: Increased generation of reactive oxygen species (ROS) in vascular diseases such as atherosclerosis, diabetes, chronic renalfailure and preeclampsia readily leads to impaired endothelium-dependent relaxation and vascular injury. To counteract ROS- andelectrophile-mediated

4、injury, cells can induce a number of genes encoding phase II detoxifying enzymes and antioxidant proteins. A cis-acting transcriptional regulatory element, designated as antioxidant response element (ARE) or electrophile response element (EpRE),mediates the transcriptional activation of genes such a

5、s heme oxygenase-1, -glutamylcysteine synthethase, thioredoxin reductase,glutathione-S-transferase and NAD(P)H:quinone oxidoreductase. Other antioxidant enzymes such as superoxide dismutase and cata-lase and non-enzymatic scavengers such as glutathione are also involved in scavenging ROS. Nuclear fa

6、ctor-erythroid 2-related factor2 (Nrf2), a member of the Cap n Collar family of basic region-leucine zipper (bZIP) transcription factors, plays an important role inARE-mediated antioxidant gene expression. Kelch-like ECH-associated protein-1 (Keap1) normally sequesters Nrf2 in the cytoplasmin associ

7、ation with the actin cytoskeleton, but upon oxidation of cysteine residues Nrf2 dissociates from Keap1, translocates to thenucleus and binds to ARE sequences leading to transcriptional activation of antioxidant and phase II detoxifying genes. Protein kinaseC (PKC), mitogen-activated protein kinases

8、(MAPKs) and phosphotidylinositol 3-kinase (PI3K) have been implicated in the regula-tion of Nrf2/ARE signaling. We here review the evidence that the Nrf2/ARE signaling pathway plays an important role in vascularhomeostasis and the defense of endothelial and smooth muscle cells against sustained oxid

9、ative stress associated with diseases such asatherosclerosis and preeclampsia.Key words: nuclear factor-erythroid 2-related factor 2; antioxidant response element; oxidative stress; endothelial cells; vascular smoothmuscle cells; heme oxygenase; cystine transporter; nitric oxide synthase; antioxidan

10、t genes; phase II detoxifying enzymes; atherosclerosis;diabetes; preeclampsia內(nèi)皮細胞和平滑肌細胞氧化應(yīng)激時Nrf2/ARE信號通路對抗氧化基因表達的調(diào)控: 與動脈粥樣硬化和先兆子癇的關(guān)系Giovanni E. Mann1,*, Jörg Niehueser-Saran1, Alan Watson1, Ling Gao1, Tetsuro Ishii2, Patricia de Winter1,Richard C. M. Siow11皇家學(xué)院醫(yī)學(xué)院心血管分部，倫敦 SE1 9NH，英國；2筑波大學(xué)人類綜合科學(xué)

11、研究科分子和細胞生理學(xué)系，茨城 305-8577，日本Received 2007-01-08 Accepted 2007-02-28This work was supported by the British Heart Foundation, Heart Research UK, Biotechnology and Biological Sciences ResearchCouncil, Medical Research Council, Wellcome Trust, KC Wong/China Scholarship Council, and EU COST ACTION B35.*Co

12、rresponding author. Tel: +44-20-78484306; Fax: +44-20-78484306; E-mail: giovanni.mannkcl.ac.uk118Acta Physiologica Sinica, April 25, 2007, 59 (2): 117-127摘 要：動脈粥樣硬化、糖尿病、慢性腎功能衰竭和先兆子癇等血管疾病時活性氧(reactive oxygen species, ROS) 生成增加，容易導(dǎo)致內(nèi)皮依賴性血管舒張功能的損害和血管損傷，而細胞可以誘導(dǎo)多種編碼II相解毒酶和抗氧化蛋白的基因表達，從而減輕ROS和親電子物質(zhì)介導(dǎo)的細胞損傷。

13、一個被稱為抗氧化反應(yīng)元件(antioxidant response element, ARE)或親電子反應(yīng)元件(electrophile response element, EpRE)的順式轉(zhuǎn)錄調(diào)控元件，可以介導(dǎo)諸如亞鐵血紅素加氧酶1、-谷氨酰半胱氨酸合成酶、硫氧還蛋白還原酶、谷胱甘肽-S轉(zhuǎn)移酶和NAD(P)H:苯醌氧化還原酶等基因的轉(zhuǎn)錄。其他抗氧化酶，如超氧化物歧化酶、過氧化氫酶和非酶清除劑(如谷胱甘肽)等也參與ROS的清除。 轉(zhuǎn)錄因子NF-E2相關(guān)因子2 (nuclear factor-erythroid 2-related factor 2,Nrf2)是屬于Cap n Collar家族的

14、轉(zhuǎn)錄因子，具有堿性亮氨酸拉鏈(basic region-leucine zipper, bZIP)，它在ARE介導(dǎo)的抗氧化基因表達中起重要的作用。在正常情況下，Kelch樣環(huán)氧氯丙烷相關(guān)蛋白-1 (Kelch-like ECH-associated protein-1, Keap1)與Nrf2耦聯(lián)，并與肌動蛋白細胞骨架結(jié)合被錨定于胞漿，但是在半胱氨酸殘基發(fā)生氧化的情況下，Nrf2和Keap1解耦聯(lián)，進入細胞核并與ARE結(jié)合，從而激活多種抗氧化基因和II相解毒酶基因的轉(zhuǎn)錄。蛋白激酶C、絲裂原活化蛋白激酶和磷脂酰肌醇-3激酶參與Nrf2/ARE信號轉(zhuǎn)導(dǎo)的調(diào)控。本文綜述了有關(guān)Nrf2/ARE信號轉(zhuǎn)導(dǎo)

15、通路在血管穩(wěn)態(tài)和動脈硬化、先兆子癇等疾病情況下內(nèi)皮及平滑肌細胞對抗持續(xù)性氧化應(yīng)激中起的作用。關(guān)鍵詞：轉(zhuǎn)錄因子NF-E2相關(guān)因子2；抗氧化反應(yīng)元件；氧化應(yīng)激；內(nèi)皮細胞；血管平滑肌細胞；亞鐵血紅素加氧酶；胱氨酸轉(zhuǎn)運體；一氧化氮合酶；抗氧化基因；相解毒酶；動脈粥樣硬化；糖尿病；先兆子癇中圖分類號：Q463；R331.3+2Nitric oxide (NO)a mediator of endothelium-dependent relaxationIn 1980, Furchgott and Zawadzki demonstrated the de-pendence of acetylcholine-

16、induced vasodilation on an in-tact endothelium and attributed smooth muscle relaxationto endothelium-derived relaxing factor (EDRF)1. Subse-quent studies established that the properties and short half-life of EDRF were identical to those of NO, a labile, ga-seous vasodilator synthesized from the sem

17、i-essentialcationic amino acid L-arginine by endothelial nitric oxidesynthase (eNOS)2-4 . The discovery that L-arginine is thephysiological precursor for NO biosynthesis precipitatedextensive research into the role of circulating and intra-cellular L-arginine in the function of vascular cells in hea

18、lthand disease4-8. As summarized in the schematic model inFig.1, classical vasoactive agonists such as histamine,bradykinin and thrombin stimulate endothelial NO synthe-sis via an elevation in intracellular calcium (Ca2+i) andCa2+/calmodulin-dependent activation of eNOS2,3,9. Incontrast, fluid shear

19、 stress, adenosine, 2-adrenoceptoragonists, 17-estradiol and soy isoflavones stimulate phos-phorylation of eNOS, dissociation of the enzyme from themembrane protein caveolin-1 and association with the chape-rone heat shock protein 90 (Hsp90), leading to increased NOproduction independent of cytosoli

20、c Ca2+ mobilization10-14.We recently reported that feeding aged male rats a soyprotein diet, rich in isoflavones genistein and daidzein, in-creases mRNA expressions of eNOS and antioxidantenzymes, improves endothelium-dependent relaxation andlowers blood pressure in vivo15. Moreover, feeding a soyis

21、oflavone-rich diet improved agonist-stimulated release ofendothelium-derived hyperpolarizing factor (EDHF) andreduced contractile force in isolated resistance vessels16, mostlikely as a consequence of elevated basal NO synthesis15.Endothelial dysfunction in preeclampsia (PE), dia-betes and intrauter

22、ine growth retardation (IUGR)Vascular diseases such as PE, diabetes, chronic renal fail-ure and atherosclerosis are all characterized by increasedoxidative stress8,17-22, and increased production of reactiveoxygen species (ROS), such as superoxide anions (O·2)which scavenge NO, leading to the f

23、ormation of peroxy-nitirite, another damaging ROS23. Reduced availability ofthe eNOS cofactor tetrahydrobiopterin and/or substrate L-· ratherarginine leads to uncoupling of eNOS, resulting in O2than NO generation24,25.It is well recognized that impaired endothelium-depen-dent relaxation in the

24、maternal circulation is a hallmark ofPE26,27, with endothelial dysfunction most likely the con-sequence of elevated plasma lipid peroxides and generation ofROS in the vasculature19,28-30 with the under-perfused placenta,a likely source of pro-inflammatory mediators29,30. PE af-fects 3%-5% of all pre

25、gnancies and is a leading cause ofmaternal and fetal morbidity and mortality. It is normallydefined as the onset of hypertension and proteinuria after20 weeks of gestation in previously normotensive and non-proteinuric pregnant women29,30. ROS-induced damageto lipids and proteins in PE results in in

26、creased hemolysis,liver damage and low platelet count (HELLP syndrome).Endothelial dysfunction in PE may be the consequence ofdiminished NO bioavailability (secondary to oxidativedegradation) and an excess of peroxynitrite31. ElevatedGiovanni E. Mann et al: Nrf2/ARE Mediated Vascular Protection119Fi

27、g. 1. Regulation of endothelial nitric oxide synthase (eNOS) via Ca2+-dependent and Ca2+-insensitive pathways. Association of eNOS withcaveolin-1 (Cav-1) in plasma membrane caveolae maintains the enzyme in an inactive state. Ca2+ mobilizing agonists such as histamine,bradykinin or thrombin result in

28、 a Ca2+-calmodulin (CaM)-dependent dissociation of eNOS from Cav-1 and association with the chaperoneheat shock protein 90 (Hsp90), leading to post-translational phosphorylation of eNOS and synthesis of NO and L-citrulline from the cationicamino acid L-arginine. Laminar shear stress and Ca2+-indepen

29、dent agonists (e.g. adenosine, 17-estradiol, isoflavones)5,12,13 activate thephosphoinositiol 3-kinase (PI3K)/protein kinase B (Akt) and extracellular-regulated kinase 1/2 (ERK1/2) and/or AMP-activated proteinkinase (AMPK)/protein kinase A (PKA) signaling pathways, leading to eNOS phosphorylation an

30、d association with Hsp90 at basal cytosolicCa2+ levels. We have shown that generation of NO in response to adenosine activates outward K+ currents, leading to a membranehyperpolarisation which in turn stimulates uptake of L-arginine via cationic amino acid transporters (CAT)4 expressed in plasma mem

31、branecaveolae12. NO activates soluble guanylyl cyclase (sGC) in endothelial cells to increase cGMP levels, which we have used to assay NOproduction (inhibitable by NOS inhibitors such as L-NAME or L-NMMA)5,9,12,13,38. Diffusion of NO to smooth muscle cells will alsoincrease cGMP levels, modulating p

32、rotein kinase G (PKG), ion channels, cGMP-activated phosphodisterases (PDE) and vascular tone2,3.nitrite/nitrate concentrations have been detected in umbili-cal vein blood in PE32, and these authors hypothesized thatan increase in feto-placental NO production may compen-sate for diminished utero-pla

33、cental blood flow. However,in PE-affected pregnancies, eNOS expression/activity iseither unchanged, decreased or increased in placental vil-lous tissue31,33,34 and regulation of eNOS by tetrahydro-biopterin is impaired35. Expression of eNOS protein in pla-cental villous tissue decreases during norma

34、l pregnancy.Although eNOS protein levels appear to be diminished inumbilical artery endothelium in pregnancies affected byPE or IUGR34, we have not detected differences in eNOSexpression in fetal endothelial cells isolated from normal,preterm and PE pregnancies36.We have reported that PE is associat

35、ed with alterationsin Ca2+ regulation, cation permeability and NO productionin human umbilical vein endothelial cells (HUVECs)36. Ca2+influx was markedly inhibited in HUVECs derived from PEpregnancies (Fig.2A), yet paradoxically both basal and his-tamine-stimulated cGMP production (as an index of NO

36、synthesis) were elevated in PE endothelial cells (Fig.2B).Increased cGMP levels in HUVECs from PE pregnanciesmay reflect activation of soluble guanylyl cyclase (sGC)by NO and/or lipid hydroperoxides, known to be elevatedin PE19,28-30. As changes in cation permeability and cGMPaccumulation persisted

37、in culture, this implies that PE in-duces phenotypic alterations in the fetal vasculature36. Ourstudies with fetal vascular smooth muscle cells (SMCs) invitro provide further evidence of vascular dysfunction inPE37. Arachidonic acid-stimulated Ca2+signaling was120Acta Physiologica Sinica, April 25,

38、2007, 59 (2): 117-127Fig.2. Oxidative stress associated with preeclampsia (PE) modulates Ca2+ mobilization and NO production in fetal endothelial and smoothmuscle cells isolated from human umbilical cords at term. A: Human umbilical vein endothelial cells (HUVECs) from normal pregnancies werestore-d

39、epleted in Ca2+-free solution with 10 µmol/L histamine in the presence of 30 µmol/L cyclopiazonic acid (CPA, Ca2+-ATPase inhibitor)to avoid internal Ca2+ store refilling. After a brief pulse of extracellular 1 mmol/L Ca2+ was applied, the rate of rise of Ca2+, Ca2+ peak and plateaulevels,

40、and rate constant for Ca2+ removal were measured. Influx rates and efflux rate constants in normal and PE endothelial cells werecompared. means±SEM of 9 normal and 6 PE cultures. B: Basal and histamine-stimulated cGMP accumulation in HUVECs (100 µmol/L L-arginine, 0.5 mmol/L 3-isobutyl-1-m

41、ethyl-xanthine in the absence or presence of L-NAME (100 µmol/L). means±SEM of 3 normal, 4 pretermand 3 PE cell cultures. P<0.02 vs normal basal, *P<0.02 vs normal histamine, §P<0.02 vs normal histamine + L-NAME. Data replotted fromSteinert et al 36. C: Arachidonic acid (AA)

42、induced increases in intracellular Ca2+ in normal and PE umbilical artery smooth muscle cells. In thepresence of extracellular Ca2+, AA (50 µmol/L)-evoked increases in intracellular Ca2+ were significantly augmented in PE cells whereas basalCa2+ levels were not significantly different. means

43、77;SEM of 3 replicate measurements from 16 normal and 19 PE cultures. *P<0.009 vs normalcells. D: Schematic model summarizing AA-induced Ca2+ mobilization in fetal smooth muscle cells from PE pregnancies. AA is metabolizedthrough the cyclooxygenase (COX), lipoxygenase (LOX) and monooxygenase (MOX

44、) pathways. In PE, the balance swings from AAmetabolism via the COX and LOX pathways to the MOX pathway, either because COX and LOX are down-regulated or because MOX isup-regulated. Increased production of MOX metabolites in turn stimulates Ca2+ entry, which can be reversed by inhibiting the MOX pat

45、hway.Data replotted from Steinert et al37.Giovanni E. Mann et al: Nrf2/ARE Mediated Vascular Protectionmodulated differentially in fetal vascular SMCs derived fromPE pregnancies, with arachidonic acid-induced Ca2+ influxincreased significantly compared to that in normal SMCs(Fig.2C). The enhanced in

46、flux of Ca2+ in PE cells wasmimicked in normal umbilical artery SMCs by inhibition ofarachidonic acid metabolism via cyclooxygenase and/orlipoxygenase pathways (Fig.2D). Based on these findings,we concluded that the potentiation of arachidonic acid-induced Ca2+ influx was due to a monooxygenase (MOX

47、)metabolite, since inhibition of the MOX signaling pathwayby structurally dissimilar compounds inhibited the enhancedCa2+ influx in indomethacin-treated normal SMCs and inarachidonic acid-challenged PE SMCs37.We and colleagues have also described phenotypicchanges in fetal endothelial cells isolated

48、 from pregnanciesaffected by either gestational diabetes or IUGR6,38-40. En-dothelial cells isolated from gestational diabetic pregnan-cies exhibit a membrane hyperpolarization and enhancedL-arginine transport and NO synthesis which persist inculture in vitro6,38. However, it is worth noting that eN

49、OSactivity is reduced in fetal endothelial cells cultured fromIUGR pregnancies40. Increased oxidative stress in preg-nancy-related diseases may thus have important implica-tions for long-term programming of the fetal cardiovas-cular system41-43, as implied by a study in which menwhose mothers suffer

50、ed from PE were at risk of develo-ping hypertension in adulthood44.ROS as intracellular signaling molecules and acti-vators of antioxidant gene expressionUnder physiological conditions, ROS are short-lived mole-cules generated as by-products of normal aerobic metabo-lism and can modulate the intrace

51、llular signaling pathwaysinvolved in the control of vascular function45-52. Exces-sive ROS generation causes damage to membrane lipids,proteins and DNA, and impairs endothelium-dependent re-laxation53. Endothelial and smooth muscle cells can gene-rate O·2 and hydrogen peroxide (H2O2) from xanth

52、ineoxidase, peroxidases, lipoxygenase, cyclooxygenases, NOSand NAD(P)H oxidases45, with membrane associatedNAD(P)H oxidase(s) serving as a primary source of ROSin vascular diseases46-48,50-52,54.Redox systems located in the vicinity of the plasma mem-brane provide protection against damage induced b

53、y envi-ronmental oxidants. Cells have evolved antioxidant defensesincluding phase II detoxifying and antioxidant enzymes, aswell as, non-enzymatic scavengers of ROS and metal ions45.Dismutation of O·2 by cytosolic copper-zinc superoxide121dismutase (CuZnSOD), mitochondrial MnSOD and extra-cellu

54、lar CuZnSOD generates H2O2, which is converted toH2O and O2 by catalase and glutathione peroxidase. En-zymes such as NAD(P)H:quinone oxidoreductase 1(NQO1), glutathione-S-transferases (GST), -glutamylcysteine synthethase (GCS), thioredoxin reduc-tases and heme oxygenases (HO) metabolize ROS and toxi

55、ccompounds to readily exportable forms. Peroxiredoxins(Prxs) are a family of antioxidant proteins that usethioredoxin as an electron donor, scavenge H2O2 and therebyalso play a key role in cellular antioxidant defenses55,56.NQO1, a two electron quinone reductase, maintains thereduced state of ubiqui

56、nones to enhance antioxidant de-fenses57. HO-1 is a microsomal enzyme induced in oxida-tive stress to metabolise heme to biliverdin, carbon mon-oxide and iron58,59. Biliverdin is subsequently convertedby biliverdin reductase to bilirubin, an antioxidant whichcan scavenge lipid peroxyl radicals while

57、 iron is seques-tered by ferritin. Carbon monoxide has both anti-apoptoticand anti-inflammatory properties and may act as a vasodi-lator in atherogenesis when bioavailability of NO is dimini-shed due to inactivation by ROS59. Moreover, HO-1 hasbeen identified in human atherosclerotic lesions, and ade-noviral overexpression of HO-1 in rodent models of vas-cular disease protects against both atherogenesis andrestenosis60-62.Transcriptional activation of antioxidant genes bythe nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) s