摘要
目的:
第二部分:为了探讨褪黑素(MEL)对血管单层内皮细胞屏障的保护作用,以培养在Transwell系统内的单层人脐静脉内皮细胞(HUVECs)为模型,观察MEL对IL-1β诱导的单层内皮细胞通透性的影响。
第二部分:观察MEL对IL-1β诱导的HUVECs间黏附连接和细胞骨架的影响。
第三部分:探讨MEL对单层内皮细胞屏障保护作用可能的受体途径和信号通路。
方法:
第一部分:原代分离培养HUVECs,并使用包被小鼠抗人CD31单克隆抗体的免疫磁珠对原代分离的HUVECs进一步纯化。以小鼠抗人vWF单克隆抗体及FITC标记的山羊抗小鼠荧光二抗鉴定纯化后的HUVECs。将纯化后的HUVECs接种在Transwell系统,通过检测不同干预条件下透过单层内皮的荧光标记的葡聚糖来反映内皮通透性的改变。
第二部分:用免疫荧光显微镜和激光共聚焦显微镜观察不同干预条件下内皮细胞黏附连接蛋白VE钙粘素(VE-cadherin)和细胞骨架蛋白的变化情况。使用Western blot检测VE-cadherin的表达变化情况。
第三部分:检测MEL和IL-1p对影响内皮细胞间连接和细胞骨架的重要的信号通路Rho GTPase家族中Rac蛋白的影响;应用MEL受体阻滞剂Luz、Rac的抑制剂NSC-23766,探讨MEL发挥HUVECs屏障保护作用的受体途径和信号通路。
结果:
第二部分:(1)成功分离培养原代HUVECs,免疫磁珠筛选后能继续贴壁生长并传代,倒置显微镜下HUVECs呈鹅卵石状,vWF免疫荧光染色证实细胞确为HUVECs。细胞呈梭形、圆形或多角形。(2)与空白对照组相比,5、10和20ng/mL的IL-1β分别使HUVECs通透性由1.5±0.1升高为4.8±0.3、5.2±0.4和9.8±0.8(P<0.01);10和100μmol/L的MEL分别使10ng/mL的IL-1β诱导的通透性从5.2±0.4降至2.8±0.3和2.7±0.3(P<0.01)。此结果与“时间-过程”通透性实验结果一致。
第三部分:(1)荧光显微镜观察:正常融合后的内皮细胞表达VE-cadherin呈现出连续不间断的状态;IL-1β(10ng/ml)破坏了细胞间的黏附连接,使VE-cadherin在细胞膜上的表达下调,并出现断裂。MEL(10μmol/L)预处理后,VE-cadherin的表达状态得到了改善。(2)激光共聚焦显微镜下观察:IL-1β(10ng/ml)明显的破坏了细胞间的黏附连接,使正常状态下分布在细胞边缘的肌动蛋白转变为跨细胞体的应力纤维,原本融合的细胞间出现明显的缝隙。MEL(10μmol/L)预处理后,没有出现明显的黏附连破坏现象,细胞中心应力纤维的表达减少,边缘部位的肌动蛋白增加,细胞间缝隙变小。(3)Western Blot结果表明:与对照组相比,IL-1p使HUVECs的VE-cadherin表达量下调。MEL可抑制IL-18诱导的VE-cadherin下调,但仍低于对照组。
第三部分:(1)Luz阻断了MEL对内皮屏障的保护作用;阻断了MEL对内皮黏附连接和细胞骨架的影响,导致细胞连接破坏和应力纤维增加,细胞间出现缝隙。(2)MEL预处理抑制了IL-1β诱导的Rac的失活,而Luz可以阻断MEL的这种作用。(3)NSC-23766抑制了MEL对内皮屏障的保护作用。
结论:(1)IL-1β诱导HUVECs黏附连接蛋白VE-cadherin表达降低,应力纤维增加,细胞间出现缝隙,通透性升高。(2)MEL通过激活Rac信号途径改善了内皮细胞黏附连接和细胞骨架的状态,抑制了IL-1β诱导的内皮通透性的升高。
Objecive
Part one:To determine the effect of melatonin (MEL) on interleukin-1β(IL-1β)-induced impairment of permeability from human umbilical vein endothelial cells (HUVECs). Part two:To determine the effect of MEL on IL-1β-induced damage of endothelial adherens junctions and actin remodeling.
Part three:To explore the possible receptors and cell signalling pathways by which melatonin exerts endothelial barrier protective effects.
Methods
Part one:Primary HUVECs were isolated from fresh human umbilical veins and cultured after immunomagnetic separation by anti-CD31 antibody coated Dynabeads. For identification of endothelial cells, the cells were incubated with mouse anti-von Willebrand Facto (vWF) and FITC-conjugated goat anti-mouse secondary antibody. The signals were detected by fluorescence microscope. Paracellular permeability was studied in a Transwell system by measuring the flux of FITC-dextran across the endothelial monolayer.
Part two:To detect actin and VE-cadherin,the cells were incubated with FITC-conjugated phalloidin and anti-VE-cadherin antibody, then observed by fluorescence microscope and confocal imaging system.
Part three:Using Rac activation assays to test the involvement of small GTPase Rac in the melatonin-induced endothelial barrier protective effects as well as cell contact reorganization. Luzindole, MEL membrane receptor antagonist and NSC-23766, a specific inhibitor to Rac were used to explore the possible receptors and cell signalling pathways.
Results
Part one:(1) Primary HUVECs were isolated sucessfully from fresh human umbilical veins and cultured after purified by immunomagnetic separation. The endothelial origin was confirmed by the positive labeling of vWF with immunofluorescence staining. (2) Treatment with IL-1β(5,10 and 20ng/ml) increased the relative permeability of HUVECs from 1.5±0.1 to 4.8±0.3,5.2±0.4 and 9.8±0.8 (P<0.01); MEL (10 and 100μmol/L) attenuated IL-1β(10ng/ml) induced increase of permeability from 5.2±0.4 to 2.8±0.3 and 2.7±0.3 (P<0.01). This result is consistent with the time course analyses of cell permeability.
Part two:(1) Fluorescence microscope:By VE-cadherin staining, treatement with IL-1βcaused a clear disruption of adherens junctions. Obvious signs of disruption of adherens junctions after the addition of melatonin in advance were not found. (2) Confocal microscope:A clear change of the actin cytoskeleton is observed from filamentous actin at the cell periphery in normal cells to stress fibers spanning the cell body in the IL-1β-induced cells. Pretreatment with melatonin induced significant reduction in central stress fibers compared with IL-1βtreated cells. (3) Western blotting: The protein level of VE-cadherin was reduced in IL-1β-treated cells, while that of the reduction was attenuated by melatonin.
Part three:(1) Luzindole effectively inhibited the protective effect of melatonin on IL-1β-induced increase of endothelial permeability. (2) The inactivation responses of the Rac in HUVECs were investigated by stimulation with IL-1β. Melatonin pretreatment inhibited the IL-1β-induced decrease in the GTPbound form of Rac. Luzindole prevented the inhibitory effects of melatonin on IL-1β-induced Rac inactivation. (3) NSC-23766 abolished the effects of melatonin on IL-1β-induced adherens junctions, cytoskeletal remodeling and the expression of VE-cadherin.
Conclusions:Our results provide evidence that L-1βtreatment disrupted HUVECs adherens junctions and induced stress fiber and paracellular gap formation. These changes increased the permeability of the endothelial monolayer. Melatonin inhibits IL-1β-induced endothelial permeability by tightening intercellular junctions and reducing IL-1β-induced stress fiber and paracellular gap formation via activation of Rac.
引文
[1]Feng Y, Venema VJ, Venema RC, et al. Vegf-induced permeability increase is mediated by caveolae [J]. Invest Ophthalmol Vis Sci,1999,40(1):157-167.
[2]Feng D, Nagy JA, Hipp J, et al. Vesiculo-vacuolar organelles and the regulation of venule permeability to macromolecules by vascular permeability factor, histamine, and serotonin [J]. J Exp Med,1996,183(5):1981-1986.
[3]Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor [J]. J Cell Sci,1995,108 (Pt 6):2369-2379.
[4]Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability [J]. Physiol Rev,2006,86(1):279-367.
[5]Wallez Y, Huber P. Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis [J]. Biochim Biophys Acta,2008,1778(3):794-809.
[6]Wojciak-Stothard B, Ridley AJ. Rho gtpases and the regulation of endothelial permeability [J]. Vascul Pharmacol,2002,39(4-5):187-199.
[7]Benz PM, Blume C, Moebius J, et al. Cytoskeleton assembly at endothelial cell-cell contacts is regulated by alphaii-spectrin-vasp complexes[J]. J Cell Biol,2008,180(1): 205-219.
[8]Birukova AA, Adyshev D, Gorshkov B, et al. Gef-h1 is involved in agonist-induced human pulmonary endothelial barrier dysfunction[J]. Am J Physiol Lung Cell Mol Physiol,2006,290(3):L540-548.
[9]Dvorak HF, Brown LF, Detmar M, et al. Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis [J]. Am J Pathol,1995,146(5):1029-1039.
[10]Roberts KA, Rezai AA, Pinkerton KE, et al. Effect of environmental tobacco smoke on ldl accumulation in the artery wall [J]. Circulation,1996,94(9):2248-2253.
[11]Juul K, Nielsen LB, Munkholm K, et al. Oxidation of plasma low-density lipoprotein accelerates its accumulation and degradation in the arterial wall in vivo [J]. Circulation,1996,94(7):1698-1704.
[12]Rangaswamy S, Penn MS, Saidel GM, et al. Exogenous oxidized low-density lipoprotein injures and alters the barrier function of endothelium in rats in vivo [J]. Circ Res,1997,80(1):37-44.
[13]Senger DR, Galli SJ, Dvorak AM, et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid [J]. Science,1983,219(4587): 983-985.
[14]Nakahara T, Norberg SM, Shalinsky DR, et al. Effect of inhibition of vascular endothelial growth factor signaling on distribution of extravasated antibodies in tumors[J]. Cancer Res,2006,66(3):1434-1445.
[15]Menon C, Ghartey A, Canter R, et al. Tumor necrosis factor-alpha damages tumor blood vessel integrity by targeting ve-cadherin [J]. Ann Surg,2006,244(5):781-791.
[16]Reyes-Aldasoro CC, Wilson I, Prise VE, et al. Estimation of apparent tumor vascular permeability from multiphoton fluorescence microscopic images of p22 rat sarcomas in vivo[J]. Microcirculation,2008,15(1):65-79.
[17]Miles FL, Pruitt FL, van Golen KL, et al. Stepping out of the flow:Capillary extravasation in cancer metastasis [J]. Clin Exp Metastasis,2008,25(4):305-324.
[18]Lee JM, Zhai G, Liu Q, et al. Vascular permeability precedes spontaneous intracerebral hemorrhage in stroke-prone spontaneously hypertensive rats [J]. Stroke, 2007,38(12):3289-3291.
[19]Lin K, Kazmi KS, Law M, et al. Measuring elevated microvascular permeability and predicting hemorrhagic transformation in acute ischemic stroke using first-pass dynamic perfusion ct imaging [J]. AJNR Am J Neuroradiol,2007,28(7):1292-1298.
[20]Grillon E, Provent P, Montigon O. et al. Blood-brain barrier permeability to manganese and to gd-dota in a rat model of transient cerebral ischaemia [J]. NMR Biomed,2008,21(5):427-436.
[21]Paul R, Zhang ZG, Eliceiri BP, et al. Src deficiency or blockade of src activity in mice provides cerebral protection following stroke [J]. Nat Med,2001,7(2):222-227.
[22]Vinores SA. Pegaptanib in the treatment of wet, age-related macular degeneration [J]. Int J Nanomedicine,2006,1(3):263-268.
[23]Lerner AB, Case JD, Takahashi Y. Isolation of melatonin and 5-methoxyindole-3-acetic acid from bovine pineal glands [J]. J Biol Chem,1960,235: 1992-1997.
[24]Silva SO, Rodrigues MR, Carvalho SR, et al. Oxidation of melatonin and its catabolites, nl-acetyl-n2 -formyl-5-methoxykynuramine and nl-acetyl-5-methoxykynuramine, by activated leukocytes [J]. J Pineal Res,2004, 37(3):171-175.
[25]Arslan SO, Gelir E, Sayan H, et al. L-arginine and melatonin interaction in rat intestinal ischemia--reperfusion [J]. Fundam Clin Pharmacol,2005,19(5):533-535.
[26]d'Emmanuele di Villa Bianca R, Marzocco S, Di Paola R, et al. Melatonin prevents lipopolysaccharide-induced hyporeactivity in rat [J]. J Pineal Res,2004,36(3): 146-154.
[27]Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria [J]. J Clin Invest,1973,52(11):2745-2756.
[28]Simko F, Paulis L. Melatonin as a potential antihypertensive treatment [J]. J Pineal Res,2007,42(4):319-322.
[29]Reiter RJ, Tan DX, Korkmaz A. The circadian melatonin rhythm and its modulation: Possible impact on hypertension [J]. J Hypertens Suppl,2009,27(6):S17-20.
[30]Zanoboni A, Zanoboni-Muciaccia W. Experimental hypertension in pinealectomized rats [J]. Life Sci,1967,6(21):2327-2331.
[31]Holmes SW, Sugden D. Proceedings:The effect of melatonin on pinealectomy-induced hypertension in the rat [J]. Br J Pharmacol,1976,56(3): 360P-361P.
[32]Staessen JA, Wang JG, Thijs L. Cardiovascular protection and blood pressure reduction:A meta-analysis [J]. Lancet,2001,358(9290):1305-1315.
[33]Staessen JA, Thijs L, Fagard R, et al. Predicting cardiovascular risk using conventional vs ambulatory blood pressure in older patients with systolic hypertension. Systolic hypertension in europe trial investigators [J]. JAMA,1999, 282(6):539-546.
[34]Xia CM, Shao CH, Xin L, et al. Effects of melatonin on blood pressure in stress-induced hypertension in rats [J]. Clin Exp Pharmacol Physiol,2008,35(10): 1258-1264.
[35]Stasica P, Ulanski P, Rosiak JM. Melatonin as a hydroxyl radical scavenger [J]. J Pineal Res,1998,25(1):65-66.
[36]Tan DX, Manchester LC, Terron MP, et al. One molecule, many derivatives:A never-ending interaction of melatonin with reactive oxygen and nitrogen species? [J]. J Pineal Res,2007,42(1):28-42.
[37]Peyrot F, Ducrocq C. Potential role of tryptophan derivatives in stress responses characterized by the generation of reactive oxygen and nitrogen species [J]. J Pineal Res,2008,45(3):235-246.
[38]Reiter RJ. Melatonin:Lowering the high price of free radicals [J]. News Physiol Sci, 2000,15:246-250.
[39]Hardeland R. Melatonin, hormone of darkness and more:Occurrence, control mechanisms, actions and bioactive metabolites [J]. Cell Mol Life Sci,2008,65(13): 2001-2018.
[40]Landmesser U, Harrison DG. Oxidant stress as a marker for cardiovascular events: Ox marks the spot [J]. Circulation,2001,104(22):2638-2640.
[41]Witztum JL, Steinberg D. The oxidative modification hypothesis of atherosclerosis: Does it hold for humans? [J]. Trends Cardiovasc Med,2001,11(3-4):93-102.
[42]Pieri C, Marra M, Gaspar R, et al. Melatonin protects ldl from oxidation but does not prevent the apolipoprotein derivatization [J]. Biochem Biophys Res Commun,1996, 222(2):256-260.
[43]Kelly MR, Loo G. Melatonin inhibits oxidative modification of human low-density lipoprotein [J]. J Pineal Res,1997,22(4):203-209.
[44]Dominguez-Rodriguez A, Abreu-Gonzalez P, Garcia-Gonzalez MJ, et al. Light/dark patterns of soluble vascular cell adhesion molecule-1 in relation to melatonin in patients with st-segment elevation myocardial infarction [J]. J Pineal Res,2008,44(1): 65-69.
[45]Carrillo-Vico A, Guerrero JM, Lardone PJ, et al. A review of the multiple actions of melatonin on the immune system [J]. Endocrine,2005,27(2):189-200.
[46]Kaur C, Sivakumar V, Lu J, et al. Melatonin attenuates hypoxia-induced ultrastructural changes and increased vascular permeability in the developing hippocampus [J]. Brain Pathol,2008,18(4):533-547.
[47]Lotufo CM, Yamashita CE, Farsky SH, et al. Melatonin effect on endothelial cells reduces vascular permeability increase induced by leukotriene b4 [J]. Eur J Pharmacol,2006,534(1-3):258-263.
[48]Thorlacius H, Lindbom L, Raud J. Cytokine-induced leukocyte rolling in mouse cremaster muscle arterioles in p-selectin dependent [J]. Am J Physiol,1997,272(4 Pt 2):H1725-1729.
[49]Eriksson EE, Werr J, Guo Y, et al. Direct observations in vivo on the role of endothelial selectins and alpha(4) integrin in cytokine-induced leukocyte-endothelium interactions in the mouse aorta [J]. Circ Res,2000,86(5): 526-533.
[50]Bhagat K, Vallance P. Inflammatory cytokines impair endothelium-dependent dilatation in human veins in vivo [J]. Circulation,1997,96(9):3042-3047.
[51]Charo IF, Taubman MB. Chemokines in the pathogenesis of vascular disease [J]. Circ Res,2004,95(9):858-866.
[1]Wang N, Butler JP, Ingber DE. Mechanotransduction across the cell surface and through the cytoskeleton [J]. Science,1993,260(5111):1124-1127.
[2]Mikuni-Takagaki Y. Mechanical responses and signal transduction pathways in stretched osteocytes [J]. J Bone Miner Metab,1999,17(1):57-60.
[3]Ivanov D, Philippova M, Antropova J, et al. Expression of cell adhesion molecule t-cadherin in the human vasculature[J]. Histochem Cell Biol,2001,115(3):231-242.
[4]Lampugnani MG, Resnati M, Raiteri M, et al. A novel endothelial-specific membrane protein is a marker of cell-cell contacts [J]. J Cell Biol,1992,118(6): 1511-1522.
[5]Bazzoni G, Dejana E. Endothelial cell-to-cell junctions:Molecular organization and role in vascular homeostasis [J]. Physiol Rev,2004,84(3):869-901.
[6]Lampugnani MG, Corada M, Caveda L, et al. The molecular organization of endothelial cell to cell junctions:Differential association of plakoglobin, beta-catenin, and alpha-catenin with vascular endothelial cadherin (ve-cadherin) [J].J Cell Biol, 1995,129(1):203-217.
[7]Panorchan P, George JP, Wirtz D. Probing intercellular interactions between vascular endothelial cadherin pairs at single-molecule resolution and in living cells[J]. J Mol Biol,2006,358(3):665-674.
[8]Legrand P, Bibert S, Jaquinod M, et al. Self-assembly of the vascular endothelial cadherin ectodomain in a ca2+-dependent hexameric structure[J]. J Biol Chem,2001, 276(5):3581-3588.
[9]Yamada S, Pokutta S, Drees F, et al. Deconstructing the cadherin-catenin-actin complex [J]. Cell,2005,123(5):889-901.
[10]Weis WI, Nelson WJ. Re-solving the cadherin-catenin-actin conundrum [J]. J Biol Chem,2006,281(47):35593-35597.
[11]Dudek SM, Garcia JG. Cytoskeletal regulation of pulmonary vascular permeability [J]. J Appl Physiol,2001,91(4):1487-1500.
[12]Navarro P, Caveda L, Breviario F, et al. Catenin-dependent and -independent functions of vascular endothelial cadherin [J]. J Biol Chem,1995,270(52): 30965-30972.
[13]Navarro P, Ruco L, Dejana E. Differential localization of ve- and n-cadherins in human endothelial cells:Ve-cadherin competes with n-cadherin for junctional localization [J]. J Cell Biol,1998,140(6):1475-1484.
[14]Gerhardt H, Wolburg H, Redies C. N-cadherin mediates pericytic-endothelial interaction during brain angiogenesis in the chicken [J]. Dev Dyn,2000,218(3): 472-479.
[15]Paik JH, Skoura A, Chae SS, et al. Sphingosine 1-phosphate receptor regulation of n-cadherin mediates vascular stabilization[J]. Genes Dev,2004,18(19):2392-2403.
[16]Lampugnani MG, Corada M, Andriopoulou P, et al. Cell confluence regulates tyrosine phosphorylation of adherens junction components in endothelial cells [J]. J Cell Sci,1997,110 (Pt 17):2065-2077.
[17]Weis SM, Cheresh DA. Pathophysiological consequences of vegf-induced vascular permeability [J]. Nature,2005,437(7058):497-504.
[18]Baumeister U, Funke R, Ebnet K, et al. Association of csk to ve-cadherin and inhibition of cell proliferation [J]. EMBO J,2005,24(9):1686-1695.
[19]Allingham MJ, van Buul JD, Burridge K. Icam-1-mediated, src- and pyk2-dependent vascular endothelial cadherin tyrosine phosphorylation is required for leukocyte transendothelial migration [J]. J Immunol,2007,179(6):4053-4064.
[20]Nawroth R, Poell G, Ranft A, et al. Ve-ptp and ve-cadherin ectodomains interact to facilitate regulation of phosphorylation and cell contacts [J]. EMBO J,2002,21(18): 4885-4895.
[21]Esser S, Lampugnani MG, Corada M, et al. Vascular endothelial growth factor induces ve-cadherin tyrosine phosphorylation in endothelial cells [J]. J Cell Sci,1998, 111 (Pt 13):1853-1865.
[22]Corada M, Mariotti M, Thurston G, et al. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo [J]. Proc Natl Acad Sci U S A,1999,96(17):9815-9820.
[23]Turowski P, Martinelli R, Crawford R, et al. Phosphorylation of vascular endothelial cadherin controls lymphocyte emigration [J]. J Cell Sci,2008,121(Pt 1):29-37.
[24]Herren B, Levkau B, Raines EW, et al. Cleavage of beta-catenin and plakoglobin and shedding of ve-cadherin during endothelial apoptosis:Evidence for a role for caspases and metalloproteinases [J]. Mol Biol Cell,1998,9(6):1589-1601.
[25]Luplertlop N, Misse D, Bray D, et al. Dengue-virus-infected dendritic cells trigger vascular leakage through metalloproteinase overproduction [J]. EMBO Rep,2006, 7(11):1176-1181.
[26]Xiao K, Allison DF, Kottke MD, et al. Mechanisms of ve-cadherin processing and degradation in microvascular endothelial cells [J]. J Biol Chem,2003,278(21): 19199-19208.
[27]Xiao K, Garner J, Buckley KM, et al. P120-catenin regulates clathrin-dependent endocytosis of ve-cadherin [J]. Mol Biol Cell,2005,16(11):5141-5151.
[28]Mehta D, Malik AB. Signaling mechanisms regulating endothelial permeability [J]. Physiol Rev,2006,86(1):279-367.
[29]Schnittler HJ. Structural and functional aspects of intercellular junctions in vascular endothelium [J]. Basic Res Cardiol,1998,93 Suppl 3:30-39.
[30]van Hinsbergh VW, van Nieuw Amerongen GP. Endothelial hyperpermeability in vascular leakage [J]. Vascul Pharmacol,2002,39(4-5):171-172.
[31]Andriopoulou P, Navarro P, Zanetti A, et al. Histamine induces tyrosine phosphorylation of endothelial cell-to-cell adherens junctions [J]. Arterioscler Thromb Vasc Biol,1999,19(10):2286-2297.
[32]Sims JR. Karp S, Ingber DE. Altering the cellular mechanical force balance results in integrated changes in cell, cytoskeletal and nuclear shape [J]. J Cell Sci,1992,103 (Pt 4):1215-1222.
[33]Meazzini MC, Toma CD, Schaffer JL, et al. Osteoblast cytoskeletal modulation in response to mechanical strain in vitro [J]. J Orthop Res,1998,16(2):170-180.
[34]Liebner S, Cattelino A, Gallini R, et al. Beta-catenin is required for endothelial-mesenchymal transformation during heart cushion development in the mouse[J]. J Cell Biol,2004,166(3):359-367.
[35]Cattelino A, Liebner S, Gallini R, et al. The conditional inactivation of the beta-catenin gene in endothelial cells causes a defective vascular pattern and increased vascular fragility [J]. J Cell Biol,2003,162(6):1111-1122.
[36]Huber AH, Weis WI. The structure of the beta-catenin/e-cadherin complex and the molecular basis of diverse ligand recognition by beta-catenin [J]. Cell,2001,105(3): 391-402.
[37]Lilien J, Balsamo J. The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin [J]. Curr Opin Cell Biol,2005, 17(5):459-465.
[38]Molecular cell biology of the endothelium in development and diseases. Abstracts of the 4th symposium on the biology of endothelial cells. Munich, germany. July 18-20, 2003 [J]. Angiogenesis,2002,5(4):281-354.
[39]Furuse M, Tsukita S. Claudins in occluding junctions of humans and flies [J]. Trends Cell Biol,2006,16(4):181-188.
[40]Morita K, Sasaki H, Furuse K, et al. Expression of claudin-5 in dermal vascular endothelia [J]. Exp Dermatol,2003,12(3):289-295.
[41]Nitta T, Hata M, Gotoh S, et al. Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice [J]. J Cell Biol,2003,161(3):653-660.
[42]Lievano S, Alarcon L, Chavez-Munguia B, et al. Endothelia of term human placentae display diminished expression of tight junction proteins during preeclampsia [J]. Cell Tissue Res,2006,324(3):433-448.
[43]Enerson BE, Drewes LR. The rat blood-brain barrier transcriptome [J]. J Cereb Blood Flow Metab,2006,26(7):959-973.
[44]Bazzoni G. Endothelial tight junctions:Permeable barriers of the vessel wall [J]. Thromb Haemost,2006,95(1):36-42.
[45]Liebner S, Fischmann A, Rascher G, et al. Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme [J]. Acta Neuropathol,2000,100(3):323-331.
[46]Davies DC. Blood-brain barrier breakdown in septic encephalopathy and brain tumours [J]. J Anat,2002,200(6):639-646.
[47]Antonetti DA, Barber AJ, Khin S, et al. Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content:Vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn state retina research group [J]. Diabetes,1998,47(12):1953-1959.
[48]Erickson KK, Sundstrom JM, Antonetti DA. Vascular permeability in ocular disease and the role of tight junctions [J]. Angiogenesis,2007,10(2):103-117.
[49]Huber JD, Egleton RD, Davis TP. Molecular physiology and pathophysiology of tight junctions in the blood-brain barrier [J]. Trends Neurosci,2001,24(12):719-725.
[50]Vajkoczy P, Menger MD. Vascular microenvironment in gliomas [J]. J Neurooncol, 2000,50(1-2):99-108.
[51]Brown RC, Davis TP. Calcium modulation of adherens and tight junction function:A potential mechanism for blood-brain barrier disruption after stroke [J]. Stroke,2002, 33(6):1706-1711.
[52]Brown RC, Davis TP. Hypoxia/aglycemia alters expression of occludin and actin in brain endothelial cells [J]. Biochem Biophys Res Commun,2005,327(4): 1114-1123.
[53]Reijerkerk A, Kooij G, van der Pol SM, et al. Diapedesis of monocytes is associated with mmp-mediated occludin disappearance in brain endothelial cells [J]. FASEB J, 2006,20(14):2550-2552.
[54]Wachtel M, Frei K, Ehler E, et al. Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition [J]. J Cell Sci,1999,112 (Pt 23):4347-4356.
[55]Antonetti DA, Wolpert EB, DeMaio L, et al. Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin [J]. J Neurochem,2002,80(4):667-677.
[56]Muller WA. Leukocyte-endothelial-cell interactions in leukocyte transmigration and the inflammatory response [J]. Trends Immunol,2003,24(6):327-334.
[57]Liang TW, DeMarco RA, Mrsny RJ, et al. Characterization of hujam:Evidence for involvement in cell-cell contact and tight junction regulation [J]. Am J Physiol Cell Physiol,2000,279(6):C1733-1743.
[58]Liu Y, Nusrat A, Schnell FJ, et al. Human junction adhesion molecule regulates tight junction resealing in epithelia [J]. J Cell Sci,2000,113 (Pt 13):2363-2374.
[59]Martin-Padura I, Lostaglio S, Schneemann M, et al. Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration [J]. J Cell Biol,1998,142(1): 117-127.
[60]Aurrand-Lions M, Johnson-Leger C, Wong C, et al. Heterogeneity of endothelial junctions is reflected by differential expression and specific subcellular localization of the three jam family members [J]. Blood,2001,98(13):3699-3707.
[61]Orlova VV, Economopoulou M, Lupu F, et al. Junctional adhesion molecule-c regulates vascular endothelial permeability by modulating ve-cadherin-mediated cell-cell contacts [J]. J Exp Med,2006,203(12):2703-2714.
[1]Naji L, Carrillo-Vico A, Guerrero JM, et al. Expression of membrane and nuclear melatonin receptors in mouse peripheral organs [J]. Life Sci,2004,74(18): 2227-2236.
[2]Dubocovich ML, Yun K, Al-Ghoul WM, et al. Selective mt2 melatonin receptor antagonists block melatonin-mediated phase advances of circadian rhythms [J]. FASEB J,1998,12(12):1211-1220.
[3]Liu C, Weaver DR, Jin X, et al. Molecular dissection of two distinct actions of melatonin on the suprachiasmatic circadian clock [J]. Neuron,1997,19(1):91-102.
[4]Reppert SM, Weaver DR, Rivkees SA, et al. Putative melatonin receptors in a human biological clock [J]. Science,1988,242(4875):78-81.
[5]Laitinen JT, Viswanathan M, Vakkuri O, et al. Differential regulation of the rat melatonin receptors:Selective age-associated decline and lack of melatonin-induced changes [J]. Endocrinology,1992,130(4):2139-2144.
[6]Weaver DR, Stehle JH, Stopa EG, et al. Melatonin receptors in human hypothalamus and pituitary:Implications for circadian and reproductive responses to melatonin [J]. J Clin Endocrinol Metab,1993,76(2):295-301.
[7]Peschke E, Fauteck JD, Musshoff U, et al. Evidence for a melatonin receptor within pancreatic islets of neonate rats:Functional, autoradiographic, and molecular investigations [J]. J Pineal Res,2000,28(3):156-164.
[8]Richter HG, Torres-Farfan C, Rojas-Garcia PP, et al. The circadian timing system: Making sense of day/night gene expression [J]. Biol Res,2004,37(1):11-28.
[9]Fujieda H, Scher J, Hamadanizadeh SA, et al. Dopaminergic and gabaergic amacrine cells are direct targets of melatonin:Immunocytochemical study of mt1 melatonin receptor in guinea pig retina [J]. Vis Neurosci,2000,17(1):63-70.
[10]Savaskan E, Wirz-Justice A, Olivieri G, et al. Distribution of melatonin mtl receptor immunoreactivity in human retina [J]. J Histochem Cytochem,2002,50(4):519-526.
[11]Meyer P, Pache M, Loeffler KU, et al. Melatonin mt-1-receptor immunoreactivity in the human eye [J]. Br J Ophthalmol,2002,86(9):1053-1057.
[12]Savaskan E, Olivieri G, Meier F, et al. Increased melatonin la-receptor immunoreactivity in the hippocampus of alzheimer's disease patients [J]. J Pineal Res, 2002,32(1):59-62.
[13]Clemens JW, Jarzynka MJ, Witt-Enderby PA. Down-regulation of mtl melatonin receptors in rat ovary following estrogen exposure [J]. Life Sci,2001,69(1):27-35.
[14]Carrillo-Vico A, Garcia-Perganeda A, Naji L, et al. Expression of membrane and nuclear melatonin receptor mrna and protein in the mouse immune system [J]. Cell Mol Life Sci,2003,60(10):2272-2278.
[15]Becker-Andre M, Wiesenberg I, Schaeren-Wiemers N, et al. Pineal gland hormone melatonin binds and activates an orphan of the nuclear receptor superfamily [J]. J Biol Chem,1994,269(46):28531-28534.
[16]Shen Q, Wu MH, Yuan SY. Endothelial contractile cytoskeleton and microvascular permeability [J]. Cell Health Cytoskelet,2009,2009(1):43-50.
[17]Ridley AJ. Rho family proteins:Coordinating cell responses [J]. Trends Cell Biol, 2001,11(12):471-477.
[18]van Nieuw Amerongen GP, van Hinsbergh VW. Cytoskeletal effects of rho-like small guanine nucleotide-binding proteins in the vascular system [J]. Arterioscler Thromb Vase Biol,2001,21(3):300-311.
[19]Ridley AJ, Hall A. The small gtp-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors [J]. Cell,1992,70(3): 389-399.
[20]Nobes CD, Hall A. Rho, rac and cdc42 gtpases:Regulators of actin structures, cell adhesion and motility [J]. Biochem Soc Trans,1995,23(3):456-459.
[21]Wojciak-Stothard B, Entwistle A, Garg R, et al. Regulation of tnf-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by rho, rac, and cdc42 in human endothelial cells [J]. J Cell Physiol,1998,176(1):150-165.
[22]Van Aelst L, D'Souza-Schorey C. Rho gtpases and signaling networks [J]. Genes Dev, 1997,11(18):2295-2322.
[23]Hall A. Rho gtpases and the actin cytoskeleton [J]. Science,1998,279(5350): 509-514.
[24]Sah VP, Seasholtz TM, Sagi SA, et al. The role of rho in g protein-coupled receptor signal transduction [J]. Annu Rev Pharmacol Toxicol,2000,40:459-489.
[25]Lampugnani MG, Zanetti A, Breviario F, et al. Ve-cadherin regulates endothelial actin activating rac and increasing membrane association of tiam [J]. Mol Biol Cell, 2002,13(4):1175-1189.
[26]Kovacs EM, Ali RG, McCormack AJ, et al. E-cadherin homophilic ligation directly signals through rac and phosphatidylinositol 3-kinase to regulate adhesive contacts [J]. J Biol Chem,2002,277(8):6708-6718.
[27]Essler M, Hermann K, Amano M, et al. Pasteurella multocida toxin increases endothelial permeability via rho kinase and myosin light chain phosphatase [J]. J Immunol,1998,161(10):5640-5646.
[28]Goeckeler ZM, Masaracchia RA, Zeng Q, et al. Phosphorylation of myosin light chain kinase by p21-activated kinase pak2 [J]. J Biol Chem,2000,275(24): 18366-18374.
[29]van Leeuwen FN, van Delft S, Kain HE, et al. Rac regulates phosphorylation of the myosin-ⅱ heavy chain, actinomyosin disassembly and cell spreading [J]. Nat Cell Biol,1999,1(4):242-248.
[30]Kolodney MS, Wysolmerski RB. Isometric contraction by fibroblasts and endothelial cells in tissue culture:A quantitative study [J]. J Cell Biol,1992,117(1):73-82.
[31]Goeckeler ZM, Wysolmerski RB. Myosin light chain kinase-regulated endothelial cell contraction:The relationship between isometric tension, actin polymerization, and myosin phosphorylation [J]. J Cell Biol,1995,130(3):613-627.
[32]Vouret-Craviari V, Boquet P, Pouyssegur J, et al. Regulation of the actin cytoskeleton by thrombin in human endothelial cells:Role of rho proteins in endothelial barrier function [J]. Mol Biol Cell,1998,9(9):2639-2653.
[33]van Nieuw Amerongen GP, Vermeer MA, van Hinsbergh VW. Role of rhoa and rho kinase in lysophosphatidic acid-induced endothelial barrier dysfunction [J]. Arterioscler Thromb Vasc Biol,2000,20(12):E127-133.
[34]van Nieuw Amerongen GP, van Delft S, Vermeer MA, et al. Activation of rhoa by thrombin in endothelial hyperpermeability:Role of rho kinase and protein tyrosine kinases[J]. Circ Res,2000,87(4):335-340.
[35]Wojciak-Stothard B, Potempa S, Eichholtz T, et al. Rho and rac but not cdc42 regulate endothelial cell permeability [J]. J Cell Sci,2001,114(Pt 7):1343-1355.
[36]Vouret-Craviari V, Bourcier C, Boulter E, et al. Distinct signals via rho gtpases and src drive shape changes by thrombin and sphingosine-1-phosphate in endothelial cells [J]. J Cell Sci,2002,115(Pt 12):2475-2484.
[37]Carbajal JM, Schaeffer RC, Jr. Rhoa inactivation enhances endothelial barrier function [J]. Am J Physiol,1999,277(5 Pt 1):C955-964.
[38]Rabiet MJ, Plantier JL, Rival Y, et al. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization [J]. Arterioscler Thromb Vasc Biol,1996,16(3):488-496.
[39]Mehta D, Rahman A, Malik AB. Protein kinase c-alpha signals rho-guanine nucleotide dissociation inhibitor phosphorylation and rho activation and regulates the endothelial cell barrier function[J]. J Biol Chem,2001,276(25):22614-22620.
[40]Aepfelbacher M, Essler M. Disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators:A role for rho/rho kinase [J]. Cell Microbiol,2001, 3(10):649-658.
[41]Hirase T, Kawashima S, Wong EY, et al. Regulation of tight junction permeability and occludin phosphorylation by rhoa-p160rock-dependent and -independent mechanisms [J]. J Biol Chem,2001,276(13):10423-10431.
[42]Minnear FL, Patil S, Bell D, et al. Platelet lipid(s) bound to albumin increases endothelial electrical resistance:Mimicked by lpa [J]. Am J Physiol Lung Cell Mol Physiol,2001,281(6):L1337-1344.
[43]Danner RL, Elin RJ, Hosseini JM, et al. Endotoxemia in human septic shock.1991 [J]. Chest,2009,136(5 Suppl):e30.
[44]Essler M, Staddon JM, Weber PC, et al. Cyclic amp blocks bacterial lipopolysaccharide-induced myosin light chain phosphorylation in endothelial cells through inhibition of rho/rho kinase signaling [J]. J Immunol,2000,164(12): 6543-6549.
[45]Breslin JW. Rock and camp promote lymphatic endothelial cell barrier integrity and modulate histamine and thrombin-induced barrier dysfunction [J]. Lymphat Res Biol, 2011,9(1):3-11.
[46]van Hinsbergh VW, van Nieuw Amerongen GP. Intracellular signalling involved in modulating human endothelial barrier function [J]. J Anat,2002,200(6):549-560.
[47]van Nieuw Amerongen GP, Draijer R, Vermeer MA, et al. Transient and prolonged increase in endothelial permeability induced by histamine and thrombin:Role of protein kinases, calcium, and rhoa [J]. Circ Res,1998,83(11):1115-1123.
[48]Vouret-Craviari V, Grall D, Flatau G, et al. Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and ve-cadherin localization in human endothelial cell monolayers[J]. Infect Immun,1999,67(6):3002-3008.
[49]Dejana E, Lampugnani MG, Martinez-Estrada O, et al. The molecular organization of endothelial junctions and their functional role in vascular morphogenesis and permeability [J]. Int J Dev Biol,2000,44(6):743-748.
[50]Corada M, Mariotti M, Thurston G, et al. Vascular endothelial-cadherin is an important determinant of microvascular integrity in vivo [J]. Proc Natl Acad Sci U S A,1999,96(17):9815-9820.
[51]Noren NK, Niessen CM, Gumbiner BM, et al. Cadherin engagement regulates rho family gtpases [J]. J Biol Chem,2001,276(36):33305-33308.
[52]Gopalakrishnan S, Dunn KW, Marrs JA. Rac1, but not rhoa, signaling protects epithelial adherens junction assembly during atp depletion [J]. Am J Physiol Cell Physiol,2002,283(1):C261-272.
[53]Kuroda S, Fukata M, Kobayashi K, et al. Identification of iqgap as a putative target for the small gtpases, cdc42 and rac1 [J]. J Biol Chem,1996,271(38):23363-23367.
[54]van Wetering S, van Buul JD, Quik S, et al. Reactive oxygen species mediate rac-induced loss of cell-cell adhesion in primary human endothelial cells [J]. J Cell Sci,2002,115(Pt 9):1837-1846.
[55]Rottner K, Hall A, Small JV. Interplay between rac and rho in the control of substrate contact dynamics [J]. Curr Biol,1999,9(12):640-648.
[56]Zondag GC, Evers EE, ten Klooster JP, et al. Oncogenic ras downregulates rac activity, which leads to increased rho activity and epithelial-mesenchymal transition [J]. J Cell Biol,2000,149(4):775-782.
[57]Von Pawel-Rammingen U, Telepnev MV, Schmidt G, et al. Gap activity of the yersinia yope cytotoxin specifically targets the rho pathway:A mechanism for disruption of actin microfilament structure [J]. Mol Microbiol,2000,36(3):737-748.
[58]Andor A. Trulzsch K, Essler M, et al. Yope of yersinia, a gap for rho gtpases, selectively modulates rac-dependent actin structures in endothelial cells [J]. Cell Microbiol,2001,3(5):301-310.
[59]Garcia JG, Liu F, Verin AD, et al. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by edg-dependent cytoskeletal rearrangement [J]. J Clin Invest,2001, 108(5):689-701.
[60]Liu F, Schaphorst KL, Verin AD, et al. Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement:Potential role of glycogen synthase kinase-3beta [J]. FASEB J,2002,16(9):950-962.
[61]Laufs U, Endres M, Stagliano N, et al. Neuroprotection mediated by changes in the endothelial actin cytoskeleton [J]. J Clin Invest,2000,106(1):15-24.
[62]Takemoto M, Sun J, Hiroki J, et al. Rho-kinase mediates hypoxia-induced downregulation of endothelial nitric oxide synthase [J]. Circulation,2002,106(1): 57-62.
[63]Eto M, Barandier C, Rathgeb L, et al. Thrombin suppresses endothelial nitric oxide synthase and upregulates endothelin-converting enzyme-1 expression by distinct pathways:Role of rho/rock and mitogen-activated protein kinase [J]. Circ Res,2001, 89(7):583-590.
[64]Sander EE, ten Klooster JP, van Delft S, et al. Rac downregulates rho activity: Reciprocal balance between both gtpases determines cellular morphology and migratory behavior [J]. J Cell Biol,1999,147(5):1009-1022.
[65]Hordijk PL, Anthony E, Mul FP, et al. Vascular-endothelial-cadherin modulates endothelial monolayer permeability [J]. J Cell Sci,1999,112 (Pt 12):1915-1923.
[66]Dubocovich ML, Rivera-Bermudez MA, Gerdin MJ, et al. Molecular pharmacology, regulation and function of mammalian melatonin receptors [J]. Front Biosci,2003,8: d1093-1108.
[67]Masana MI, Doolen S, Ersahin C, et al. Mt(2) melatonin receptors are present and functional in rat caudal artery [J]. J Pharmacol Exp Ther,2002,302(3):1295-1302.
[68]Savaskan E, Olivieri G, Brydon L, et al. Cerebrovascular melatonin mt1-receptor alterations in patients with alzheimer's disease [J]. Neurosci Lett,2001,308(1):9-12.
[69]Krause DN, Barrios VE, Duckles SP. Melatonin receptors mediate potentiation of contractile responses to adrenergic nerve stimulation in rat caudal artery [J]. Eur J Pharmacol,1995,276(3):207-213.
[70]Regrigny O, Delagrange P, Scalbert E, et al. Melatonin improves cerebral circulation security margin in rats [J]. Am J Physiol,1998,275(1 Pt 2):H139-144.
[71]Doolen S, Krause DN, Dubocovich ML, et al. Melatonin mediates two distinct responses in vascular smooth muscle [J]. Eur J Pharmacol,1998,345(1):67-69.
[72]Cui P, Yu M, Luo Z, et al. Intracellular signaling pathways involved in cell growth inhibition of human umbilical vein endothelial cells by melatonin [J]. J Pineal Res, 2008,44(1):107-114.
[73]Braga VM. Cell-cell adhesion and signalling [J]. Curr Opin Cell Biol,2002,14(5): 546-556.
[74]Waschke J, Baumgartner W, Adamson RH, et al. Requirement of rac activity for maintenance of capillary endothelial barrier properties [J]. Am J Physiol Heart Circ Physiol,2004,286(1):H394-401.
[75]Sandoval R, Malik AB, Minshall RD, et al. Ca(2+) signalling and pkcalpha activate increased endothelial permeability by disassembly of ve-cadherin junctions [J]. J Physiol,2001,533(Pt 2):433-445.