oxLDL干扰HUVECs的DSG1和DSC2表达并增加单层内皮细胞对LDL的通透性
|
摘要
研究背景:关于动脉粥样硬化(AS)的发生,目前普遍认为与血浆中脂蛋白水平的升高,血管内皮细胞的损伤导致的动脉壁通透性增加以及脂蛋白穿过内皮屏障在内皮下沉积等有关。在动脉粥样硬化(AS)斑块和泡沫细胞中检测到了氧化修饰的低密度脂蛋白(oxLDL),提示oxLDL与AS的发生发展密切相关。但迄今为止未见oxLDL促进内皮细胞对LDL通透性机制方面的报道。桥粒芯糖蛋白-1(DSG1)和桥粒芯胶蛋白-2(DSC2)属于桥粒钙粘素蛋白家族成员,存在于血管内皮细胞间隙,发挥了血管壁与血液间的物理屏障作用,维持细胞与细胞之间的完整性。因此内皮细胞与细胞间的这种桥粒连接在通透性调控方面起了重要的作用。本研究意在探讨xLDL对内皮细胞DSG1和DSC2表达的影响,以及由此导致对内皮细胞通透性的影响。
实验一oxLDL对HUVEC细胞DSG1,DSC2表达的影响
目的:观察oxLDL对人脐静脉内皮细胞上跨膜蛋白DSG1和DSC2表达的影响,以及人脐静脉内皮细胞受到这种氧化型脂蛋白刺激后对单层细胞通透性的变化。
方法:每次实验前24小时,给HUVECs换新鲜培养基培养后加处理因素。不同浓度oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)处理HUVEC24h,检测细胞DSG1,DSC2 mRNA和蛋白的表达,再用50mg/L oxLDL分别处理HUVEC不同时间(0h,6h,12h,24h),用Western blotting和RT- PCR分别检测细胞DSG1,DSC2 mRNA和蛋白的表达。
结果:HUVECs上有DSG1、DSC2 mRNA与蛋白质的表达;oxLDL使DSG1、DSC2 mRNA与蛋白的表达下调,且成时间与剂量依赖关系(P<0.05)。
实验二oxLDL对HUVECs单层通透性的影响
目的:观察oxLDL对人脐静脉内皮细胞单层通透性的影响。
方法:用不同浓度的oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)分别或者与SOD共同孵育HUVECs24h,空白组作为对照,通过Transwell系统检测单层细胞对BSA和LDL的通透性情况,采用荧光分光光度法测定FITC-BSA和FITC-LDL的含量。
结果:oxLDL能显著增加HUVECs单层对BSA和LDL的通透性,且作用随着浓度增加而增强,在50mg/L时有显著作用(P<0.05);SOD能使50mg/L oxLDL诱导的HUVECs单层通透性降低(P<0.05)。
实验三ROS参与oxLDL对HUVEC DSG1和DSC2表达的调节
目的:探讨oxLDL是否通过促进细胞内ROS生成参与调节HUVEC细胞DSG1和DSC2的表达。
方法:用LDL(50mg/L)、oxLDL(50mg/L)、BSA(100mg/L)、H2O2(5mg/L)分别与HUVECs孵育24h,空白组作为对照,用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平。用50mg/L的oxLDL、50mg/LSOD预处理再加oxLDL、50mg/LSOD分别与HUVECs孵育24h,用DCFH-DA染色法检测细胞内活性氧的生成情况;用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平;用激光共聚焦显微镜观察细胞DSG1的免疫反应性。
结果:oxLDL、H2O2使HUVECs DSG1、DSC2 mRNA与蛋白质的表达下调(P<0.05),而LDL、BSA对其无明显影响(P>0.05)。oxLDL增加细胞内活性氧的生成和降低DSG1、DSC2 mRNA与蛋白质的表达(P<0.05),同时降低HUVECs DSG1免疫反应性,且SOD均能抑制上述结果。
结论:oxLDL具有干扰DSG1和DSC2的表达并增加血管内皮对LDL的通透性的作用;活性氧的产生增加可能是oxLDL所介导的单层内皮细胞通透作用的途径之一。
BACKGROUND
The mechanism of the atherosclerosis (As), is now generally believed closely associated with the increased of lipoproteins level. The increase of endothelial permeability of the arterial vessel wall is favorite for lipoprotein deposit in subendothelial space. Studies has showed that the presence of oxidatively modified low-density lipoprotein (oxLDL) detected in the intima of atherosclerotic plaques and foam cells. It is believed that the oxLDL is closely related to As. But so far,it has not been clearly understood if the oxLDL promotes the endothelium permeability to LDL. Both desmoglein1(DSG1) and desmocollin2 (DSC2) are the member of cadherins. They function at the site of interface of the endothelial cells, and play a role of the vessel walls’physical barriers and maintaining the integrity of the cell to cell. The desmosomal junction of the cell-cell in the permeability regulation may play an important role. this study is to explore the effects of oxLDL on expression of DSG1and DSC2 in endothelial cells, the effect of permeability injury induced by oxidized lipoprotein, and possible mechanisms of these.
Part I. Effects of oxLDL on DSG1,DSC2 expression in HUVECs
Objective: To identify the effects of oxLDL on DSG1 and DSC2 expression in HUVECs.
Methods: Replaced with fresh medium every 24 hours before treatments. The different concentrations of oxLDL (0 mg/L,10 mg/L,25 mg/L,50 mg/L) were incubated with HUVECs for 24h, or with 50mg/L of oxLDL for different times (0h, 6h,12h and 24h). RT-PCR and Western blotting were applied to determine the DSG1 and DSC2 expression in mRNA and protein, respectively.
Results: We found that oxLDL decreased DSG1 and DSC2 in both protein and mRNA levels in dose-dependent and time-dependent manners (P<0.05)..
Part II. Effects of oxLDL on monolayer permeability in HUVECs
Objective: To investigate the effects of oxLDL on monolayer permeability in HUVECs.
Methods: HUVECs were cultured in DMEM medium, and replaced with fresh medium every 24 hours before treatments. Microscope was used to observe the monolayer information. HUVECs cultured in upper compartment of transwell were treated by oxLDL(50mg/L) and/or SOD(50mg/L) for 24 hours. And then, The LDL was added in upper compartment for 4 hours until to test the content of the LDL in the lower compartment. This effect represented change of the monolayer permeability to LDL.
Results: The oxidized lipoprotein promoted the endothelial monolayer permeability to LDL(P<0.05). Superoxide dismutase(SOD) partly inhibited the effects of oxLDL on monolayer barrier damage(P<0.05).
PartⅢ. ROS involved in oxLDL regulation of DSG1 and DSC2 expression in HUVECs
Objective: To explore the role of ROS in the regulation of expression on DSG1 and DSC2 induced by oxLDL in HUVECs.
Methods: HUVECs were incubated with oxLDL(50mg/L) and LDL(50mg/L), BSA(100mg/L), H2O(25mg/L), respectively, for 24h. control group was designed to PBS incubation. HUVECs were pretreated for 1h with 50mg/L superoxide dismutase(SOD), and then were incubated for another 24h with 50mg/l of oxLDL. Intracellular ROS production was detected by Dichlorofluorescin diacetate (DCFH-DA) dye. The mRNA and protein expression of DSG1and DSC2 was determined by RT- PCR and Western blotting, respectively. Immunoreactivity of DSG1 was detected by laser scanning confocal microscope (LSCM).
Results: oxLDL and H2O2 decreased expression of DSG1, DSC2, both mRNA and protein, in HUVECs(P<0.05) , while detected effects were not observed in treatment groups by LDL and BSA. oxLDL increased generation of cellular reactive oxygen species, and reduced expression of DSG1, DSC2 mRNA and protein(P<0.05). Furthermore, oxLDL reduced immunostaining of DSG1 in HUVECs. The results also showed this effect was inhibited by SOD supplement.
Conclusions :
These findings suggest that oxLDL may interfere the expression of desmosome proteins, DSG1 and DSC2, by which endothelial permeability to LDL is increased. Production of reactive oxygen species(ROS) may be involved in the promotion of monolayer permeability by oxLDL.
实验一oxLDL对HUVEC细胞DSG1,DSC2表达的影响
目的:观察oxLDL对人脐静脉内皮细胞上跨膜蛋白DSG1和DSC2表达的影响,以及人脐静脉内皮细胞受到这种氧化型脂蛋白刺激后对单层细胞通透性的变化。
方法:每次实验前24小时,给HUVECs换新鲜培养基培养后加处理因素。不同浓度oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)处理HUVEC24h,检测细胞DSG1,DSC2 mRNA和蛋白的表达,再用50mg/L oxLDL分别处理HUVEC不同时间(0h,6h,12h,24h),用Western blotting和RT- PCR分别检测细胞DSG1,DSC2 mRNA和蛋白的表达。
结果:HUVECs上有DSG1、DSC2 mRNA与蛋白质的表达;oxLDL使DSG1、DSC2 mRNA与蛋白的表达下调,且成时间与剂量依赖关系(P<0.05)。
实验二oxLDL对HUVECs单层通透性的影响
目的:观察oxLDL对人脐静脉内皮细胞单层通透性的影响。
方法:用不同浓度的oxLDL(0 mg/L,10 mg/L,25 mg/L,50 mg/L)分别或者与SOD共同孵育HUVECs24h,空白组作为对照,通过Transwell系统检测单层细胞对BSA和LDL的通透性情况,采用荧光分光光度法测定FITC-BSA和FITC-LDL的含量。
结果:oxLDL能显著增加HUVECs单层对BSA和LDL的通透性,且作用随着浓度增加而增强,在50mg/L时有显著作用(P<0.05);SOD能使50mg/L oxLDL诱导的HUVECs单层通透性降低(P<0.05)。
实验三ROS参与oxLDL对HUVEC DSG1和DSC2表达的调节
目的:探讨oxLDL是否通过促进细胞内ROS生成参与调节HUVEC细胞DSG1和DSC2的表达。
方法:用LDL(50mg/L)、oxLDL(50mg/L)、BSA(100mg/L)、H2O2(5mg/L)分别与HUVECs孵育24h,空白组作为对照,用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平。用50mg/L的oxLDL、50mg/LSOD预处理再加oxLDL、50mg/LSOD分别与HUVECs孵育24h,用DCFH-DA染色法检测细胞内活性氧的生成情况;用RT-PCR与Western blotting分别检测HUVECs DSG1、DSC2 mRNA与蛋白质的表达水平;用激光共聚焦显微镜观察细胞DSG1的免疫反应性。
结果:oxLDL、H2O2使HUVECs DSG1、DSC2 mRNA与蛋白质的表达下调(P<0.05),而LDL、BSA对其无明显影响(P>0.05)。oxLDL增加细胞内活性氧的生成和降低DSG1、DSC2 mRNA与蛋白质的表达(P<0.05),同时降低HUVECs DSG1免疫反应性,且SOD均能抑制上述结果。
结论:oxLDL具有干扰DSG1和DSC2的表达并增加血管内皮对LDL的通透性的作用;活性氧的产生增加可能是oxLDL所介导的单层内皮细胞通透作用的途径之一。
BACKGROUND
The mechanism of the atherosclerosis (As), is now generally believed closely associated with the increased of lipoproteins level. The increase of endothelial permeability of the arterial vessel wall is favorite for lipoprotein deposit in subendothelial space. Studies has showed that the presence of oxidatively modified low-density lipoprotein (oxLDL) detected in the intima of atherosclerotic plaques and foam cells. It is believed that the oxLDL is closely related to As. But so far,it has not been clearly understood if the oxLDL promotes the endothelium permeability to LDL. Both desmoglein1(DSG1) and desmocollin2 (DSC2) are the member of cadherins. They function at the site of interface of the endothelial cells, and play a role of the vessel walls’physical barriers and maintaining the integrity of the cell to cell. The desmosomal junction of the cell-cell in the permeability regulation may play an important role. this study is to explore the effects of oxLDL on expression of DSG1and DSC2 in endothelial cells, the effect of permeability injury induced by oxidized lipoprotein, and possible mechanisms of these.
Part I. Effects of oxLDL on DSG1,DSC2 expression in HUVECs
Objective: To identify the effects of oxLDL on DSG1 and DSC2 expression in HUVECs.
Methods: Replaced with fresh medium every 24 hours before treatments. The different concentrations of oxLDL (0 mg/L,10 mg/L,25 mg/L,50 mg/L) were incubated with HUVECs for 24h, or with 50mg/L of oxLDL for different times (0h, 6h,12h and 24h). RT-PCR and Western blotting were applied to determine the DSG1 and DSC2 expression in mRNA and protein, respectively.
Results: We found that oxLDL decreased DSG1 and DSC2 in both protein and mRNA levels in dose-dependent and time-dependent manners (P<0.05)..
Part II. Effects of oxLDL on monolayer permeability in HUVECs
Objective: To investigate the effects of oxLDL on monolayer permeability in HUVECs.
Methods: HUVECs were cultured in DMEM medium, and replaced with fresh medium every 24 hours before treatments. Microscope was used to observe the monolayer information. HUVECs cultured in upper compartment of transwell were treated by oxLDL(50mg/L) and/or SOD(50mg/L) for 24 hours. And then, The LDL was added in upper compartment for 4 hours until to test the content of the LDL in the lower compartment. This effect represented change of the monolayer permeability to LDL.
Results: The oxidized lipoprotein promoted the endothelial monolayer permeability to LDL(P<0.05). Superoxide dismutase(SOD) partly inhibited the effects of oxLDL on monolayer barrier damage(P<0.05).
PartⅢ. ROS involved in oxLDL regulation of DSG1 and DSC2 expression in HUVECs
Objective: To explore the role of ROS in the regulation of expression on DSG1 and DSC2 induced by oxLDL in HUVECs.
Methods: HUVECs were incubated with oxLDL(50mg/L) and LDL(50mg/L), BSA(100mg/L), H2O(25mg/L), respectively, for 24h. control group was designed to PBS incubation. HUVECs were pretreated for 1h with 50mg/L superoxide dismutase(SOD), and then were incubated for another 24h with 50mg/l of oxLDL. Intracellular ROS production was detected by Dichlorofluorescin diacetate (DCFH-DA) dye. The mRNA and protein expression of DSG1and DSC2 was determined by RT- PCR and Western blotting, respectively. Immunoreactivity of DSG1 was detected by laser scanning confocal microscope (LSCM).
Results: oxLDL and H2O2 decreased expression of DSG1, DSC2, both mRNA and protein, in HUVECs(P<0.05) , while detected effects were not observed in treatment groups by LDL and BSA. oxLDL increased generation of cellular reactive oxygen species, and reduced expression of DSG1, DSC2 mRNA and protein(P<0.05). Furthermore, oxLDL reduced immunostaining of DSG1 in HUVECs. The results also showed this effect was inhibited by SOD supplement.
Conclusions :
These findings suggest that oxLDL may interfere the expression of desmosome proteins, DSG1 and DSC2, by which endothelial permeability to LDL is increased. Production of reactive oxygen species(ROS) may be involved in the promotion of monolayer permeability by oxLDL.
引文
[1] Harrison D, Griendling KK, Landmesser U, et al.Role of oxidative stress in atherosclerosis[J]. Am J Cardiol, 2003,91: 7A–11A.
[2] Lassegue B, Griendling KK. Reactive oxygen species in hypertension:an update[J]. Am J Hypertens, 2004, 17: 852–860.
[3] Vincent AM, Russell JW, Low P, et al. Oxidative stress in the pathogenesis of diabetic neuropathy[J]. Endocr Rev, 2004,25: 612–628 .
[4] Landmesser U, Harrison DG. Oxidant stress as a marker for cardiovascular events: Ox marks the spot[J] .Circulation,2001, 104: 2638–2640 .
[5] Steinberg D,Witztum JL. Is the oxidative modification hypothesis relevant to human atherosclerosis? : Do the antioxidant trials conducted to date refute the hypothesis?[J] .Circulation ,2002,105: 2107–2111 .
[6] Hayashida K, Kume N, Minami M, et al. Lectin-like oxidized LDL receptor-1 (LOX-1) supports adhesion of mononuclear leukocytes and a monocyte-like cell line THP-1 cells under static and flow conditions[J] .FEBS Lett, 2002,511: 133–138 .
[7] Kaplan M, Aviram M. Retention of oxidized LDL by extracellular matrix proteoglycans leads to its uptake by macrophages: an alternative approach to study lipoproteins cellular uptake[J] .Arterioscler Thromb Vasc Biol, 2001,21: 386–393 .
[8] Wada Y, Sugiyama A, Kohro T, et al. In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage[J]. Yonsei Med J, 2000,41: 740–755 .
[9] Dimmeler S, Haendeler J, Galle J, et al. Oxidized low density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the“response to injury”hypothesis[J] .Circulation,1997, 95: 1760–1763 .
[10] Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins causecontraction and inhibit endothelium-dependent relaxation in the pig coronary artery[J]. J Clin Invest ,86: 75–79, 1990.
[11] Bochkov VN, Kadl A, Huber J, et al. Protective role of phospholipid oxidation products in endotoxininduced tissue damage[J] .Nature, 2002,419: 77–81 .
[12] Furnkranz A, Schober A, Bochkov VN,et al. Oxidized phospholipids trigger atherogenic inflammation in murine arteries[J]. Arterioscler Thromb Vasc Biol,2005, 25: 633–638 .
[13] Reddy ST, Grijalva V, Ng C, et al. Identification of genes induced by oxidized phospholipids in human aortic endothelial cells[J]. Vasc Pharmacol, 2002,38: 211–218.
[14] Hulthe J, Fagerberg B. Circulating Oxidized LDL Is Associated With Subclinical Atherosclerosis Development and Inflammatory Cytokines (AIR Study)[J]. Arterioscler Thromb Vasc Biol, 2002, 22: 1162 - 1167.
[15] Ziouzenkova O, Asatryan L, Akmal M, et al. Oxidative crosslinking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis[J].J Biol Chem,1999,274: 18916–18924 .
[16] Hwang J, Ing MH, Salazar A, et al. Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit expression: implication for native LDL oxidation[J].Circ Res,2003, 93: 1225–1232 .
[17] Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease[J] .Arterioscler Thromb Vasc Biol ,2005,25: 29–38 .
[18] Akisaka T, Yoshida H, Suzuki R, et al.Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture[J].Cell Tissue Res, 2008,331(3): 625-41.
[19] Jennifer M, Halbleib W, Nelson J.Cadherins in development: cell adhesion, sorting, and tissue morphogenesis [J]. Genes & Dev, 2006, 20: 3199 - 3214 .
[20] Gumbiner BM.Cell adhension:the molecular basis of tissue architecture and morphogenesis[J] .Cell ,1996,84:345-35733.
[21] Dejana E, Corada M, Lampugnani MG.Endothelial cell-to-cell junctions[J].FASEB ,1995,9:910-8
[22] Elisabetta D.Perspectives Series:Cell adhesion in vascular biology-Endothelial adherins junctions:implications in the control of vascular permeability and angiogenesis[J]. J Clin Invest, 1996,98:1949-1953
[23] Peifer M.Cell adhension and signal transduction:the Armadillo connection[J]. Trends Cell Biol,1995,5:224-9.
[24] Hiraki A,Shinohara M,Ikebe T,et al.Immunohistochemical staining of desmosomal components in oral squamous cell carcinomas and its association with tumour behaviour[J].Br J Cancer, 1996,73(12): 1491-7.
[25] Silberberg M, Audra J.Bacallao R,et al.Mispolarization of desmosomal proteins and altered intercellular adhesion in autosomal dominant polycystic kidney disease [J].Am J Physiol Renal Physiol, Jun 2005, 288: F1153 - F1163.
[26] Wang W, Dentler WL, Borchardt RT. VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly[J] Am J Physiol Heart Circ Physiol ,2001,280: H434–H440.
[27] Antonetti DA, Barber AJ, Hollinger LA, et al. Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors[J].J Biol Chem, 1999,274: 23463–23467 .
[28] Melissa L, Min Y, Yan L, et al. Transactivation of Vascular Endothelial Growth Factor Receptor-2 by Interleukin-8 (IL-8/CXCL8) Is Required for IL-8/CXCL8- induced Endothelial Permeability[J]. Mol. Biol. Cell, Dec 2007,18: 5014 - 5023.
[29] DeMaio L, Chang YS, Gardner TW, et al.Shear stress regulates occludin content and phosphorylation[J]. Am J Physiol Heart Circ Physiol, 2001,281: H105–H113 .
[30] Feldman GJ, Mullin JM, Ryan MP. Occludin: structure, function and regulation[J] .Adv Drug Delivery Res,2005 ,57: 883–917 .
[31] Wang Y, Chang J, Li YC, et al. Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway[J]. Am J Physiol Heart Circ Physiol ,2004, 286: H685–H692.
[32] Kuzuya M, Ramos MA, Kanda S, et al. VEGF protects against oxidized LDL toxicity to endothelial cells by an intracellular glutathione-dependent mechanism through the KDR receptor[J] .Arterioscler Thromb Vasc Biol, 2001,21:765–770 .
[33] Wang JF, Zhang X, Groopman JE. Activation of vascular endothelial growth factor receptor-3 and its downstream signaling promote cell survival under oxidative stress[J] .J Biol Chem, 2004,279: 27088–27097 .
[34] Denu JM ,Tanner KG. Redox regulation of protein tyrosine phosphatases by hydrogen peroxide: detecting sulfenic acid intermediates and examining reversible inactivation[J] .Methods Enzymol ,2002,348: 297–305 .
[35] Birukov KG, Leitinger N, Bochkov VN, et al. Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins[J] .Microvasc Res,2004, 67: 18–28.
[36] Traweger A, Fang D, Liu YC, et al. The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase Itch[J].J Biol Chem, 2002, 277:10201–10208 .
[37] Holthofer B, Windoffer R, Troyanovsky S.Structure and function of desmosomes.[J].Int Rev Cytol, 2007, 264: 65-163.
[38] Pasdar M, Li Z, Chan H.Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells[J].Cell Motil Cytoskeleton, 1995,30(2): 108-21.
[39] Amar LS, Shabana al-HM, Oboeuf M, et al.Desmosomes are regulated by protein kinase C in primary rat epithelial cells[J].Cell Adhes Commun, 1998 ,5(1): 1-12.
[40] DeMaioL, Rouhanizadeh M, Reddy S, et al.Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells[J].Am J Physiol Heart Circ Physiol ,2006,290: H674–H683 .
[1]王迪浔,金惠铭.人体病理生理学[M ].北京:北京人民卫生出版社, 2002:515-516.
[2] Cyrus T ,Pratico D ,Zhao L , et al . Absence of 12/ 15 - lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein E-deficient mice[J]. Circulation ,2001 ,103 (18) :2277-2282.
[3] Molavi B,Mehta JL. Oxidative stress in cardiovascular disease:Molecular basis of its deleterious effects, its detection, and therapeutic considerations [J]. Curr Op in Cardiol, 2004, 19 (5) : 488-493.
[4]吴蕊,涂玲.氧化应激与心血管疾病[J].心血管病学进展,2007,28(1):110-113.
[5] Heitzer T, Schlinzig T, Krohn K, et al. Endothelial dysfunction, oxidative stress,and risk of cardiovascular events in patients with coronary artery disease [J].Circulation, 2001, 104: 2673-2678.
[6] Sorescu GP, Song H, Tressel SL, et al. Bonemorphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase [J]. Circ Res, 2004, 95 (8) : 773-779
[7] Lin SJ , Shyue SK, Liu PL, et al. Adenovirus-mediated overexpression of catalase attenuates oxLDL-induced apoptosis in human aortic endothelial cells via AP-1 and C-Jun N-terminal kinase/extracellular signal-regulated kinase mitogen- activated protein kinase pathways [J]. J Mol Cell Cardiol, 2004, 36 (1) : 129-139.
[8] Xia Z, LiuM, Wu Y, et al. N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression[J]. Eur J Pharmacol, 2006, 550 (123) : 134-142.
[9] B runt KR, Fenrich KK, Kiani G, et al. Protection of human vascular smooth muscle cells from H2O2-induced apop tosis through functional codependence between HO-1 and AKT [J]. A rterioscler Thromb Vasc Biol,2006, 26 (9) : 2 027-034.
[10]陈瑗,周玫.氧化应激-炎症在动脉粥样硬化发生发展中作用研究的新进展[J].中国动脉硬化杂志,2008,16(10):757-762.
[11] Pueyo ME,Gonzalez W,Nicoletti A,et al[J].Arterioscler Thromb Vasc Biol,2000,20(3):645-651.
[12] Denger S,Jahn L,Wende P,et al, Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis. [J].Atherosclerosis,1999,144(1):15-23.
[13] Ramos MA,Kuzuya M,Esaki T,et al, Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions [J].Arterioscler Thromb Vasc Biol,1998,18(7):1188-1196.
[14] Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature molecular and cellularmechanisms [J]. Hypertension, 2003, 42: 1075-1081.
[15] Zhang H, Chalothorn D, Jackson LF, et al. Transactivation of epidermal growth factor receptor mediates catecholamine-induced growth of vascular smooth muscle [J]. Circ Res, 2004, 95 (10) : 989-997.
[16] Chai YC,Howe PH,DiCorlto PE,et al. Oxidized Low Density Lipoprotein and Lysophosphatidylcholine Stimulate Cell Cycle Entry in Vascular Smooth Muscle Cells[J].J Biol Chem,1996,271(30):17791-17797.
[17] Roberts CP,Parthasarathy S,Gulati R,et al. Effect of RU-486 and related compounds on the proliferation of cultured macrophages[J].Am J Reprod Immunol,1995,34:248-256.
[18] Augs N,Andrieu N,Negre-Salvayre A,et al. The Sphingomyelin-Ceramide Signaling Pathway Is Involved in Oxidized Low Density Lipoprotein-induced Cell Proliferation [J].J Biol Chem,1996,271(32):19251-19255.
[19] Petit L,Lesnik P,Dachet C,et al. Tissue Factor Pathway Inhibitor Is Expressed by Human Monocyte–Derived Macrophages:Relationship to Tissue Factor Induction by Cholesterol and Oxidized LDL [J].Arteioscler Thromb Vasc Biol,1999, 19(2),309-315.
[20] Florea SM, B latterLA. The effect of oxidative stress on Ca2 + release and capacitative Ca2 + entry in vascular endothelial cells [J]. Cell Calcium,2008, 43(4) : 405-415.
[21] Besse S, Bulteau AL, Boucher F, et al. Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improvesmyocardial function and coronary perfusion in senescent hearts [J]. J Physiol Pharm acol, 2006, 57 (4) : 541-552.
[22] Pajovic SB, Radojcic MB, Kanazir DT. Neuroendocrine and oxidoreductive mechanisms of stress-induced cardiovascular diseases[J]. Physiol Res,2008, 57 (3) : 327-338.
[23] Bianca J. J,Brunde M, Shiroshita-Takeshita A, et al. Induction of heat shock response p rotects the heart against atrial fibrillation [J]. Circ Res,2006, 99 (12) : 1394-402.
[24] Suzuki A, Lu J , Kusakai G, et al. ARK5 is a tumor invasion-associated factor downstream ofAkt signaling [J]. M ol Cell B iol, 2004, 24 (8) :3526-3535.
[25] Donald D. Heistad.Oxidative Stress and Vascular Disease 2005 Duff Lecture[J].Arterioscler Thromb Vasc Biol, 2006,26:689-695.
[26] Faraci FM, Didion SP. Vascular protection: Superoxide dismutase isoforms in the vessel wall [J].Arterioscler Thromb Vasc Biol,2004,24:1367–1373.
[27] MacMillan-Crow LA, Cruthirds DL. Manganese superoxide dismutase in disease[J].Free Radic Res, 2001,34:325–337.
[28]‘t Hoen PAC, Van der Lans CAC, Van Eck M, Bijsterbosch MK, Van Berkel TJC, Twisk J. Aorta of apoE-deficient mice responds to atherogenic stimuli by a pre-lesional increase and subsequent decrease in the expression of antioxidant enzymes[J]. Circ Res, 2003,93:262–269.
[29] Daiber A, Oelze M, Sulyok S, et al. Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance[J]. Mol. Pharmacol, 2005,68: 579 - 588.
[30] Yang H, Roberts LJ, Shi M-J, et al. Retardation of atherosclerosis by overexpression of catalase or both Cu/ZnSOD dismutase and catalase in mice lacking apolipoprotein E [J].Circ es,2004,95: 1075–1081.
[31] Oury TD, Day BJ, Crapo JD. Extracellular superoxide dismutase: A regulator of nitric oxide bioavailability [J].Lab Invest, 1996,75:617– 636.
[32]林燕,黄维义,李著华.氧化应激致动脉粥样硬化作用的研究进展[J].黑龙江医学,2004,28(7):517-519.
[33]李震霄,邹洪梅,孟晓萍.氧化应激促进动脉粥样硬化机制研究进展[J].中国动脉硬化杂志, 2009, 17 (8): 702-705.
[2] Lassegue B, Griendling KK. Reactive oxygen species in hypertension:an update[J]. Am J Hypertens, 2004, 17: 852–860.
[3] Vincent AM, Russell JW, Low P, et al. Oxidative stress in the pathogenesis of diabetic neuropathy[J]. Endocr Rev, 2004,25: 612–628 .
[4] Landmesser U, Harrison DG. Oxidant stress as a marker for cardiovascular events: Ox marks the spot[J] .Circulation,2001, 104: 2638–2640 .
[5] Steinberg D,Witztum JL. Is the oxidative modification hypothesis relevant to human atherosclerosis? : Do the antioxidant trials conducted to date refute the hypothesis?[J] .Circulation ,2002,105: 2107–2111 .
[6] Hayashida K, Kume N, Minami M, et al. Lectin-like oxidized LDL receptor-1 (LOX-1) supports adhesion of mononuclear leukocytes and a monocyte-like cell line THP-1 cells under static and flow conditions[J] .FEBS Lett, 2002,511: 133–138 .
[7] Kaplan M, Aviram M. Retention of oxidized LDL by extracellular matrix proteoglycans leads to its uptake by macrophages: an alternative approach to study lipoproteins cellular uptake[J] .Arterioscler Thromb Vasc Biol, 2001,21: 386–393 .
[8] Wada Y, Sugiyama A, Kohro T, et al. In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage[J]. Yonsei Med J, 2000,41: 740–755 .
[9] Dimmeler S, Haendeler J, Galle J, et al. Oxidized low density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the“response to injury”hypothesis[J] .Circulation,1997, 95: 1760–1763 .
[10] Simon BC, Cunningham LD, Cohen RA. Oxidized low density lipoproteins causecontraction and inhibit endothelium-dependent relaxation in the pig coronary artery[J]. J Clin Invest ,86: 75–79, 1990.
[11] Bochkov VN, Kadl A, Huber J, et al. Protective role of phospholipid oxidation products in endotoxininduced tissue damage[J] .Nature, 2002,419: 77–81 .
[12] Furnkranz A, Schober A, Bochkov VN,et al. Oxidized phospholipids trigger atherogenic inflammation in murine arteries[J]. Arterioscler Thromb Vasc Biol,2005, 25: 633–638 .
[13] Reddy ST, Grijalva V, Ng C, et al. Identification of genes induced by oxidized phospholipids in human aortic endothelial cells[J]. Vasc Pharmacol, 2002,38: 211–218.
[14] Hulthe J, Fagerberg B. Circulating Oxidized LDL Is Associated With Subclinical Atherosclerosis Development and Inflammatory Cytokines (AIR Study)[J]. Arterioscler Thromb Vasc Biol, 2002, 22: 1162 - 1167.
[15] Ziouzenkova O, Asatryan L, Akmal M, et al. Oxidative crosslinking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis[J].J Biol Chem,1999,274: 18916–18924 .
[16] Hwang J, Ing MH, Salazar A, et al. Pulsatile versus oscillatory shear stress regulates NADPH oxidase subunit expression: implication for native LDL oxidation[J].Circ Res,2003, 93: 1225–1232 .
[17] Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease[J] .Arterioscler Thromb Vasc Biol ,2005,25: 29–38 .
[18] Akisaka T, Yoshida H, Suzuki R, et al.Adhesion structures and their cytoskeleton-membrane interactions at podosomes of osteoclasts in culture[J].Cell Tissue Res, 2008,331(3): 625-41.
[19] Jennifer M, Halbleib W, Nelson J.Cadherins in development: cell adhesion, sorting, and tissue morphogenesis [J]. Genes & Dev, 2006, 20: 3199 - 3214 .
[20] Gumbiner BM.Cell adhension:the molecular basis of tissue architecture and morphogenesis[J] .Cell ,1996,84:345-35733.
[21] Dejana E, Corada M, Lampugnani MG.Endothelial cell-to-cell junctions[J].FASEB ,1995,9:910-8
[22] Elisabetta D.Perspectives Series:Cell adhesion in vascular biology-Endothelial adherins junctions:implications in the control of vascular permeability and angiogenesis[J]. J Clin Invest, 1996,98:1949-1953
[23] Peifer M.Cell adhension and signal transduction:the Armadillo connection[J]. Trends Cell Biol,1995,5:224-9.
[24] Hiraki A,Shinohara M,Ikebe T,et al.Immunohistochemical staining of desmosomal components in oral squamous cell carcinomas and its association with tumour behaviour[J].Br J Cancer, 1996,73(12): 1491-7.
[25] Silberberg M, Audra J.Bacallao R,et al.Mispolarization of desmosomal proteins and altered intercellular adhesion in autosomal dominant polycystic kidney disease [J].Am J Physiol Renal Physiol, Jun 2005, 288: F1153 - F1163.
[26] Wang W, Dentler WL, Borchardt RT. VEGF increases BMEC monolayer permeability by affecting occludin expression and tight junction assembly[J] Am J Physiol Heart Circ Physiol ,2001,280: H434–H440.
[27] Antonetti DA, Barber AJ, Hollinger LA, et al. Vascular endothelial growth factor induces rapid phosphorylation of tight junction proteins occludin and zonula occluden 1. A potential mechanism for vascular permeability in diabetic retinopathy and tumors[J].J Biol Chem, 1999,274: 23463–23467 .
[28] Melissa L, Min Y, Yan L, et al. Transactivation of Vascular Endothelial Growth Factor Receptor-2 by Interleukin-8 (IL-8/CXCL8) Is Required for IL-8/CXCL8- induced Endothelial Permeability[J]. Mol. Biol. Cell, Dec 2007,18: 5014 - 5023.
[29] DeMaio L, Chang YS, Gardner TW, et al.Shear stress regulates occludin content and phosphorylation[J]. Am J Physiol Heart Circ Physiol, 2001,281: H105–H113 .
[30] Feldman GJ, Mullin JM, Ryan MP. Occludin: structure, function and regulation[J] .Adv Drug Delivery Res,2005 ,57: 883–917 .
[31] Wang Y, Chang J, Li YC, et al. Shear stress and VEGF activate IKK via the Flk-1/Cbl/Akt signaling pathway[J]. Am J Physiol Heart Circ Physiol ,2004, 286: H685–H692.
[32] Kuzuya M, Ramos MA, Kanda S, et al. VEGF protects against oxidized LDL toxicity to endothelial cells by an intracellular glutathione-dependent mechanism through the KDR receptor[J] .Arterioscler Thromb Vasc Biol, 2001,21:765–770 .
[33] Wang JF, Zhang X, Groopman JE. Activation of vascular endothelial growth factor receptor-3 and its downstream signaling promote cell survival under oxidative stress[J] .J Biol Chem, 2004,279: 27088–27097 .
[34] Denu JM ,Tanner KG. Redox regulation of protein tyrosine phosphatases by hydrogen peroxide: detecting sulfenic acid intermediates and examining reversible inactivation[J] .Methods Enzymol ,2002,348: 297–305 .
[35] Birukov KG, Leitinger N, Bochkov VN, et al. Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins[J] .Microvasc Res,2004, 67: 18–28.
[36] Traweger A, Fang D, Liu YC, et al. The tight junction-specific protein occludin is a functional target of the E3 ubiquitin-protein ligase Itch[J].J Biol Chem, 2002, 277:10201–10208 .
[37] Holthofer B, Windoffer R, Troyanovsky S.Structure and function of desmosomes.[J].Int Rev Cytol, 2007, 264: 65-163.
[38] Pasdar M, Li Z, Chan H.Desmosome assembly and disassembly are regulated by reversible protein phosphorylation in cultured epithelial cells[J].Cell Motil Cytoskeleton, 1995,30(2): 108-21.
[39] Amar LS, Shabana al-HM, Oboeuf M, et al.Desmosomes are regulated by protein kinase C in primary rat epithelial cells[J].Cell Adhes Commun, 1998 ,5(1): 1-12.
[40] DeMaioL, Rouhanizadeh M, Reddy S, et al.Oxidized phospholipids mediate occludin expression and phosphorylation in vascular endothelial cells[J].Am J Physiol Heart Circ Physiol ,2006,290: H674–H683 .
[1]王迪浔,金惠铭.人体病理生理学[M ].北京:北京人民卫生出版社, 2002:515-516.
[2] Cyrus T ,Pratico D ,Zhao L , et al . Absence of 12/ 15 - lipoxygenase expression decreases lipid peroxidation and atherogenesis in apolipoprotein E-deficient mice[J]. Circulation ,2001 ,103 (18) :2277-2282.
[3] Molavi B,Mehta JL. Oxidative stress in cardiovascular disease:Molecular basis of its deleterious effects, its detection, and therapeutic considerations [J]. Curr Op in Cardiol, 2004, 19 (5) : 488-493.
[4]吴蕊,涂玲.氧化应激与心血管疾病[J].心血管病学进展,2007,28(1):110-113.
[5] Heitzer T, Schlinzig T, Krohn K, et al. Endothelial dysfunction, oxidative stress,and risk of cardiovascular events in patients with coronary artery disease [J].Circulation, 2001, 104: 2673-2678.
[6] Sorescu GP, Song H, Tressel SL, et al. Bonemorphogenic protein 4 produced in endothelial cells by oscillatory shear stress induces monocyte adhesion by stimulating reactive oxygen species production from a nox1-based NADPH oxidase [J]. Circ Res, 2004, 95 (8) : 773-779
[7] Lin SJ , Shyue SK, Liu PL, et al. Adenovirus-mediated overexpression of catalase attenuates oxLDL-induced apoptosis in human aortic endothelial cells via AP-1 and C-Jun N-terminal kinase/extracellular signal-regulated kinase mitogen- activated protein kinase pathways [J]. J Mol Cell Cardiol, 2004, 36 (1) : 129-139.
[8] Xia Z, LiuM, Wu Y, et al. N-acetylcysteine attenuates TNF-alpha-induced human vascular endothelial cell apoptosis and restores eNOS expression[J]. Eur J Pharmacol, 2006, 550 (123) : 134-142.
[9] B runt KR, Fenrich KK, Kiani G, et al. Protection of human vascular smooth muscle cells from H2O2-induced apop tosis through functional codependence between HO-1 and AKT [J]. A rterioscler Thromb Vasc Biol,2006, 26 (9) : 2 027-034.
[10]陈瑗,周玫.氧化应激-炎症在动脉粥样硬化发生发展中作用研究的新进展[J].中国动脉硬化杂志,2008,16(10):757-762.
[11] Pueyo ME,Gonzalez W,Nicoletti A,et al[J].Arterioscler Thromb Vasc Biol,2000,20(3):645-651.
[12] Denger S,Jahn L,Wende P,et al, Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis. [J].Atherosclerosis,1999,144(1):15-23.
[13] Ramos MA,Kuzuya M,Esaki T,et al, Induction of Macrophage VEGF in Response to Oxidized LDL and VEGF Accumulation in Human Atherosclerotic Lesions [J].Arterioscler Thromb Vasc Biol,1998,18(7):1188-1196.
[14] Taniyama Y, Griendling KK. Reactive oxygen species in the vasculature molecular and cellularmechanisms [J]. Hypertension, 2003, 42: 1075-1081.
[15] Zhang H, Chalothorn D, Jackson LF, et al. Transactivation of epidermal growth factor receptor mediates catecholamine-induced growth of vascular smooth muscle [J]. Circ Res, 2004, 95 (10) : 989-997.
[16] Chai YC,Howe PH,DiCorlto PE,et al. Oxidized Low Density Lipoprotein and Lysophosphatidylcholine Stimulate Cell Cycle Entry in Vascular Smooth Muscle Cells[J].J Biol Chem,1996,271(30):17791-17797.
[17] Roberts CP,Parthasarathy S,Gulati R,et al. Effect of RU-486 and related compounds on the proliferation of cultured macrophages[J].Am J Reprod Immunol,1995,34:248-256.
[18] Augs N,Andrieu N,Negre-Salvayre A,et al. The Sphingomyelin-Ceramide Signaling Pathway Is Involved in Oxidized Low Density Lipoprotein-induced Cell Proliferation [J].J Biol Chem,1996,271(32):19251-19255.
[19] Petit L,Lesnik P,Dachet C,et al. Tissue Factor Pathway Inhibitor Is Expressed by Human Monocyte–Derived Macrophages:Relationship to Tissue Factor Induction by Cholesterol and Oxidized LDL [J].Arteioscler Thromb Vasc Biol,1999, 19(2),309-315.
[20] Florea SM, B latterLA. The effect of oxidative stress on Ca2 + release and capacitative Ca2 + entry in vascular endothelial cells [J]. Cell Calcium,2008, 43(4) : 405-415.
[21] Besse S, Bulteau AL, Boucher F, et al. Antioxidant treatment prevents cardiac protein oxidation after ischemia-reperfusion and improvesmyocardial function and coronary perfusion in senescent hearts [J]. J Physiol Pharm acol, 2006, 57 (4) : 541-552.
[22] Pajovic SB, Radojcic MB, Kanazir DT. Neuroendocrine and oxidoreductive mechanisms of stress-induced cardiovascular diseases[J]. Physiol Res,2008, 57 (3) : 327-338.
[23] Bianca J. J,Brunde M, Shiroshita-Takeshita A, et al. Induction of heat shock response p rotects the heart against atrial fibrillation [J]. Circ Res,2006, 99 (12) : 1394-402.
[24] Suzuki A, Lu J , Kusakai G, et al. ARK5 is a tumor invasion-associated factor downstream ofAkt signaling [J]. M ol Cell B iol, 2004, 24 (8) :3526-3535.
[25] Donald D. Heistad.Oxidative Stress and Vascular Disease 2005 Duff Lecture[J].Arterioscler Thromb Vasc Biol, 2006,26:689-695.
[26] Faraci FM, Didion SP. Vascular protection: Superoxide dismutase isoforms in the vessel wall [J].Arterioscler Thromb Vasc Biol,2004,24:1367–1373.
[27] MacMillan-Crow LA, Cruthirds DL. Manganese superoxide dismutase in disease[J].Free Radic Res, 2001,34:325–337.
[28]‘t Hoen PAC, Van der Lans CAC, Van Eck M, Bijsterbosch MK, Van Berkel TJC, Twisk J. Aorta of apoE-deficient mice responds to atherogenic stimuli by a pre-lesional increase and subsequent decrease in the expression of antioxidant enzymes[J]. Circ Res, 2003,93:262–269.
[29] Daiber A, Oelze M, Sulyok S, et al. Heterozygous Deficiency of Manganese Superoxide Dismutase in Mice (Mn-SOD+/-): A Novel Approach to Assess the Role of Oxidative Stress for the Development of Nitrate Tolerance[J]. Mol. Pharmacol, 2005,68: 579 - 588.
[30] Yang H, Roberts LJ, Shi M-J, et al. Retardation of atherosclerosis by overexpression of catalase or both Cu/ZnSOD dismutase and catalase in mice lacking apolipoprotein E [J].Circ es,2004,95: 1075–1081.
[31] Oury TD, Day BJ, Crapo JD. Extracellular superoxide dismutase: A regulator of nitric oxide bioavailability [J].Lab Invest, 1996,75:617– 636.
[32]林燕,黄维义,李著华.氧化应激致动脉粥样硬化作用的研究进展[J].黑龙江医学,2004,28(7):517-519.
[33]李震霄,邹洪梅,孟晓萍.氧化应激促进动脉粥样硬化机制研究进展[J].中国动脉硬化杂志, 2009, 17 (8): 702-705.