氧化型低密度脂蛋白对培养的内皮细胞的致凋亡作用及尼莫地平的保护作用

通过研究氧化型低密度脂蛋白对培养的内皮细胞生长状态的影响及其致凋亡作用,阐述其致动脉粥样硬化机理,并研究尼莫地平对氧化型低密度脂蛋白诱导的内皮细胞凋亡的作用。用Cu^2 引发脂质过氧化过程,制备氧化型低密度脂蛋白,用MTT检测法检测细胞活性,PI杂色法及ELISA法检测细胞凋亡。结果显示,细胞生长对氧化型低密度脂蛋白呈浓度依赖性抑制作用。ELISA法和PI染色法进一步证实氧化型低密度脂蛋白可诱导内皮细胞发生凋亡。而尼莫地平可以抑制氧化型低密度脂蛋白诱导的内皮细胞凋亡。提示氧化型低密度脂蛋白对内皮细胞的细胞毒作用及其作用剂量有关;体内氧化型低密度脂蛋白通过致内皮细胞凋亡的途径,损伤血管内皮,引发动脉粥样硬化。尼莫地平可以对氧化型低密度脂蛋白诱导的内皮细胞凋亡起到保护作用。     

氧化型高密度脂蛋白对细胞凋亡及细胞内钙离子浓度的影响

目的通过研究氧化型高密度脂蛋白对培养的内皮细胞致凋亡以及细胞内钙离子浓度的影响,阐述其致动脉粥样硬化机理,并探讨其引起细胞损伤的机制.方法用铜离子引发脂质过氧化过程,制备氧化型高密度脂蛋白,用MTT检测法检测细胞活性,PI染色法检测细胞凋亡.结果氧化型高密度脂蛋白可诱导内皮细胞发生凋亡;细胞内钙离子浓度则随氧化型高密度脂蛋白浓度的升高呈现逐渐下降的趋势.结论氧化型高密度脂蛋白引起细胞损伤的机制可能与钙离子浓度升高有关,体内氧化型高密度脂蛋白通过内皮细胞凋亡的途径损伤血管内皮,引发动脉粥样硬化.     

不同浓度葡萄糖对人血管内皮细胞的影响

目的研究高浓度葡萄糖对人血管内皮细胞的影响。方法在人脐静脉内皮细胞株ECV304培养基中分别加入5.5、20、40 mmol/L葡萄糖,作用24~72 h。采用四甲基偶氮唑盐微量酶反应比色（MTT）法观察细胞增殖情况,倒置相差显微镜观察细胞形态,透射电镜观察内皮细胞V304凋亡情况;流式细胞术检测细胞凋亡率。结果高浓度葡萄糖能明显抑制血管内皮细胞的增殖,诱导培养的内皮细胞发生凋亡,细胞凋亡率增加,并随浓度的增高、时间的延长作用明显。结论高浓度葡萄糖能抑制培养的人血管内皮细胞增殖,促进细胞凋亡。     

酰基化ghrelin抑制高糖诱导的人血管内皮细胞凋亡

目的近年来研究证明高糖引起的血管内皮细胞凋亡可能加重糖尿病动脉粥样硬化,本研究旨在探讨酰基化ghrelin能否抑制高糖诱导的人血管内皮细胞凋亡。方法应用吖啶橙形态学染色及TUNEL、流式细胞分析仪、分光光度计研究高糖及ghrelin预处理后内皮细胞凋亡及caspase-3活性情况。结果内皮细胞暴露于高浓度葡萄糖(33.3mmol/L)72h与正常水平葡萄糖(5.5mmol/L)相比凋亡细胞数量显著增加,酰基化ghrelin(10-7mol/L)预处理24h后显著降低高糖诱导的凋亡。同时高糖环境下凋亡蛋白caspase-3活性增高,酰基化ghrelin预处理后明显降低caspase-3活性。结论酰基化ghrelin可以抑制高糖诱导的血管内皮细胞凋亡及凋亡蛋白caspase-3的表达,可能在防治糖尿病动脉粥样硬化的过程中起到一定作用。     

波动性高糖对血管平滑肌细胞增殖及凋亡蛋白表达的影响

目的探讨波动性高葡萄糖对血管平滑肌细胞增殖及凋亡蛋白表达的影响。方法以持续高糖和波动性高糖分别孵育大鼠胸主动脉平滑肌细胞24 h,采用MTT法检测血管平滑肌细胞增殖活性,流式细胞术检测细胞周期改变,Western bloting检测胞浆中Bcl-2、Bax蛋白的表达。结果波动性高浓度葡萄糖(5.5 mmol/L和25mmol/L交替)培养较持续高浓度葡萄糖(25 mmol/L)培养可明显增加血管平滑肌细胞增殖活性,促进其由G0/G1期向S期转变,上调Bcl-2/Bax比值。结论波动性高葡萄糖可能通过干预细胞周期及对凋亡蛋白的调控,进一步促进血管平滑肌细胞增殖,诱导其凋亡,因而较持续性高葡萄糖更能促进糖尿病动脉粥样硬化的发生发展。     

扇贝裙边糖胺聚糖拮抗高糖对血管内皮细胞损伤机理的研究

目的 探讨扇贝裙边糖胺聚糖(SS-GAG)拮抗血管内皮细胞损伤性疾病的价值.方法 取体外培养生长良好的血管内皮细胞(ECV304),建立高糖诱导的细胞损伤模型.在高浓度葡萄糖(55.5 mmol/L)状态下加入不同浓度SS-GAG,继续培养至12、24、36、48 h,倒置显微镜下观察细胞形态,用3H-TdR掺入法检测细胞增殖活性.结果 ①高浓度葡萄糖使ECV304细胞形态发生改变,细胞增殖活性受到明显抑制,且呈浓度和时间依赖性;②SS-GAG与高浓度葡萄糖共同作用下,内皮细胞形态及增殖活性基本正常,此效果与SS-GAG浓度呈正比.结论 高浓度葡萄糖对血管内皮细胞具有明显损伤作用;SS-GAG可减轻高浓度葡萄糖对血管内皮细胞的损伤,保护血管内皮细胞;SS-GAG对防治心脑血管疾病和糖尿病血管病变及动脉粥样硬化的发生、发展有积极意义.     

脂质过氧化和维生素E对培养人内皮细胞的影响

用联胺作用于培养人内皮细胞,激发并促进了脂质过氧化。随联胺浓度增加,内皮细胞过氧化脂质浓度增加,前列环素合成减少。其形态变化主要为细胞收缩,质膜泡形成,内质网扩张,线粒体肿胀,细胞空泡化及坏死脱落。抗氧化剂维生素E能显著降低联胺引起的过氧化脂质的升高,增加前列环素的合成并明显减轻内皮细胞的形态损伤。但在高浓度联胺时,维生素E无明显保护作用。这些结果提示,脂质过氧化可能通过损伤内皮细胞而在动脉粥样硬化发生中起重要作用,维生素E由于保护内皮细胞的氧化性损伤而可能有防治动脉粥样硬化的作用。     

氧化应激与动脉粥样硬化

氧化应激以及在氧化应激过程中产生的活性氧与多种疾病的发生有关,包括高血压、糖尿病、衰老、癌症和心血管疾病如动脉粥样硬化。本文阐述了氧化应激与动脉粥样硬化之间的关系。氧化应激不仅促进低密度脂蛋白的氧化修饰和脂质过氧化,而且可以诱导血管基因表达的改变和促进细胞增殖,促进动脉粥样硬化的发生。近年有新的研究认为,轻度的氧化应激对机体可能产生有益作用,这为进一步明确动脉粥样硬化的发病机制提供了新的见解。     

氯沙坦抑制低密度脂蛋白诱导大鼠血管内皮细胞凋亡及其介导机制

目的 :研究氯沙坦抑制氧化低密度脂蛋白 (oxLDL)诱导的血管内皮细胞凋亡及其介导机制。方法 :应用大鼠模型 ,低密度脂蛋白 (LDL)体内诱导血管内皮细胞凋亡 ,氯沙坦进行干预 ,观察其对LDL诱导血管内皮细胞凋亡的影响。TUNEL法检测凋亡细胞 ,比色法测caspase 3酶活性 ,SP免疫组化法分析p5 3蛋白的表达。结果 :LDL组与对照组比较血管内皮细胞凋亡数显著增加 ,caspase 3酶活性显著增高 ,p5 3蛋白表达增强 ;氯沙坦干预组与LDL组比较凋亡细胞数显著减少 ,caspase 3酶活性下降 ,p5 3蛋白表达降低。结论 :LDL可通过激活caspase 3酶及上调p5 3蛋白的表达诱导大鼠血管内皮细胞凋亡 ;氯沙坦可通过抑制caspase 3酶活性及下调p5 3蛋白表达抑制LDL诱导凋亡     

槲皮素对血管内皮细胞损伤的保护作用

目的 :研究槲皮素对血管内皮细胞损伤的保护作用及机制。　　方法 :实验分对照组、模型组和槲皮素组 ,用过氧化氢诱导血管内皮细胞损伤 ,四甲基偶氮唑蓝比色法检测细胞存活数量 ,放射免疫法测定细胞培养液中的内皮素及前列环素含量 ,用荧光法检测细胞内脂质过氧化产物丙二醛以评价脂质过氧化程度。　　结果 :槲皮素 5、2 0、40、80和 10 0 μmol/ L 促进培养的正常血管内皮细胞增殖 ,增加前列环素生成 ,减少内皮素基础释放量 ;75 μmol/ L过氧化氢可损伤培养的血管内皮细胞 ;槲皮素 5、2 0和 80 μmol/ L 可抑制过氧化氢损伤的血管内皮细胞释放乳酸脱氢酶、内皮素及促进损伤的内皮细胞释放前列环素。　　结论 :槲皮素具有保护血管内皮细胞损伤的作用 ,其机制可能与其抗脂质过氧化作用有关。     

Thiamine and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose

BACKGROUND: High glucose induces pathological alterations in small and large vessels, possibly through increased formation of AGE, activation of aldose reductase and protein kinase C, and increased flux through the hexosamine pathway. We showed previously that thiamine and benfotiamine correct delayed replication and increase lactate production in endothelial cells subjected to high glucose. We now aim at verifying the effects of thiamine and benfotiamine on cell cycle, apoptosis, and expression of adhesion molecules in endothelial cells and pericytes, under high ambient glucose. METHODS: Human umbilical vein endothelial cells and bovine retinal pericytes were cultured in normal (5.6 mmol/L) or high (28 mmol/L) glucose, with or without thiamine or benfotiamine, 50 or 100 micro mol/L. Apoptosis was determined by two separate ELISA methods, measuring DNA fragmentation and caspase-3 activity, respectively. Cell cycle and integrin subunits alpha3, alpha5, and beta1 concentration were measured by flow cytometry. RESULTS: Apoptosis was increased in high glucose after 3 days of culture, both in endothelium and pericytes. Thiamine and benfotiamine reversed such effects. Neither cell cycle traversal nor integrin concentrations were modified in these experimental conditions. CONCLUSIONS: Thiamine and benfotiamine correct increased apoptosis due to high glucose in cultured vascular cells. Further elucidations of the mechanisms through which they work could help set the basis for clinical use of this vitamin in the prevention and/or treatment of diabetic microangiopathy.     

High glucose augments stress-induced apoptosis in endothelial cells

Hyperglycemia has been identified as one of the important factors involved in the microvascular complications of diabetes, and has been related to increased cardiovascular mortality. Endothelial damage and dysfunction result from diabetes; therefore, the aim of this study was to determine the response of endothelial cells to stressful stimuli, modelled in normal and high glucose concentrations in vitro. EAhy 926 endothelial ceils were cultured in 5 mmol/L or 30 mmol/L glucose conditions for a 24 hour period and oxidative stress was induced by exposure to hydrogen peroxide （HzO2） or tumour necrosis factor- α （TNF- α ）, following which the protective effect of the glucocorticoid dexamethasone was assessed. Apoptosis, necrosis and cell viability were determined using an ELISA for DNA fragmentation, an enzymatic lactate dehydrogenase assay and an MTT assay, respectively. High glucose significantly increased the susceptibility of EAhy 926 cells to apoptosis in the presence of 500 gmol/L H2O2 , above that induced in normal glucose （P〈0.02）. A reduction of H2O2- and TNF- a -induced apoptosis occurred in both high and low glucose after treatment with dexamethasone （P〈0.05）. Conclusion high glucose is effective in significantly augmenting stress caused by H2O2, but not in causing stress alone. These findings suggest a mechanism by which short term hyperglycemia may facilitate and augment endothelial damage.     

The role of polyol pathway in high glucose-induced endothelial cell damages

To clarify the mechanism by which hyperglycemia in diabetes mellitus causes endothelial cell damages, the effects of high glucose on DNA fragmentation and caspase-3 activity of cultured endothelial cells and on the generation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were studied. Furthermore, the involvement of the polyol pathway in this process was investigated using aldose reductase inhibitor (SNK-860). Human umbilical vein endothelial cells (HUVECs) were incubated with 5.5mmol/L (low glucose medium) or 28mmol/L (high glucose medium) of glucose. The amounts of fragmented DNA, caspase-3 activity and 8-OHdG in the medium increased in significantly greater extent in high glucose-incubated HUVECs than in low glucose-incubated HUVECs. No significant increase in fragmented DNA or 8-OHdG was observed when HUVECs were incubated with mannitol (500mg/mL). The concentration of intracellular sorbitol was significantly higher in HUVECs incubated in high glucose medium than that in low glucose medium. Addition of the aldose reductase inhibitor SNK-860 dose-dependently decreased the intracellular sorbitol concentration in HUVECs incubated in high glucose medium, and also significantly suppressed the increases in fragmented DNA, caspase-3 activity and 8-OHdG by conditioning with high glucose medium. These results suggest that high glucose-induced endothelial cell damages may be mediated by activation of the polyol pathway accompanied by augmented oxidative stress.     

葡萄糖及胰岛素对牛血管内皮细胞凋亡的影响

目的通过对牛血管内皮细胞（ＥＣ）的体外培养研究糖尿病（ＤＭ）状况下葡萄糖（Ｇｌｕ）、胰岛素（Ｉｎｓ）对ＥＣ凋亡的影响。方法吖啶橙／溴乙锭（ＡＯ／ＥＢ）荧光染色及ＤＮＡ片段的琼脂糖凝胶电泳对ＥＣ凋亡定性,ＥＬＩＳＡ方法测定ＤＮＡ片段定量ＥＣ凋亡。结果高浓度Ｇｌｕ（２０ｍｍｏｌ／Ｌ、４０ｍｍｏｌ／Ｌ）和Ｉｎｓ（３００ｍＵ／Ｌ、３０００ｍＵ／Ｌ）能够诱发ＥＣ发生凋亡。ＥＣ凋亡与Ｇｌｕ和Ｉｎｓ浓度成正比,并且为时间依赖性。低浓度Ｉｎｓ（３０ｍＵ／Ｌ）明显改善Ｇｌｕ４０ｍｍｏｌ／Ｌ下所致ＥＣ的凋亡,但高浓度Ｉｎｓ与Ｇｌｕ有正性协同作用,增加ＥＣ凋亡。高浓度甘露醇（Ｍａｎ２０ｍｍｏｌ／Ｌ、４０ｍｍｏｌ／Ｌ）不引起ＥＣ明显凋亡。结论ＤＭ环境下高血糖、高Ｉｎｓ血症能诱发ＥＣ凋亡,促进ＤＭ血管并发症的发生、发展     

High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration

Delayed wound healing and accelerated atherosclerosis are common vascular complications of diabetes mellitus. Although elevated blood glucose level is the major contributing factor, mechanisms that mediate these complications are not clearly understood. In the present study, we have demonstrated that elevated glucose inhibits endothelial cell migration, thereby delaying wound healing. Our results clearly indicated that high glucose (10 or 30 mM) induced activation of nuclear factor kappa B (NF-kappaB) inhibited endothelial cell migration (P<0.05). High glucose induced NF-kappaB DNA binding activity may mediate this inhibition of migration by regulating intracellular nitric oxide. In vitro wound healing model in human aortic endothelial cells (HAEC) were used to evaluate cell migration under the influence of high glucose. The migration inhibited by high glucose was restored by NF-kappaB inhibitors (including E3-4-methylphenyl sulfonyl-2-propenenitrile, N-tosyl-Lys-chloromethylketone (TLCK), or over-expression of inhibitor subunit of kappaB) and endothelial nitric oxide synthase inhibitors (N-methyl-L-arginine (L-NMMA); and Nomega-nitro-L-arginine methyl ester (L-NAME)). Furthermore, NF-kappaB inhibitors attenuated high glucose induced eNOS expression and intracellular nitric oxide (NO) production. Cytoskeletal immunofluorescence staining confirmed differences in actin distribution in HAEC incubated in high glucose in the presence or absence of NF-kappaB and NO inhibitors, explaining the differences observed in migration. In summary, our results for the first time suggest therapeutic strategies involving inhibition of NF-kappaB activation induced by high glucose, which may improve wound healing and help avoid some of the vascular complications of diabetes.     

Oxidized low-density lipoprotein stimulates p53-dependent activation of proapoptotic Bax leading to apoptosis of differentiated endothelial progenitor cells

Dyslipidemia increases the risks for atherosclerosis in part by impairing endothelial integrity; endothelial progenitor cells (EPCs) play a pivotal role in reendothelialization. In this study, we investigated the mechanism whereby oxidized low-density lipoprotein (oxLDL) affects the function of differentiated EPCs (EDCs). In EDCs expanded in vitro from EPCs isolated from human cord blood, we measured EDC responses to both copper-oxidized LDL and L5, an electronegative LDL minimally oxidized in vivo in patients with hypercholesterolemia. OxLDL induced apoptosis of EDCs and impaired their response to nitric oxide. We found that the key to oxLDL-induced apoptosis in both EDCs and endothelial cells is the induction of a conformational change of Bax, leading to Bax activation without altering its expression. The conformationally changed Bax translocated to the mitochondria and stimulated apoptosis, as Bax knockdown prevented oxLDL-induced apoptosis in EDCs. The activation of Bax is mediated by an increase in p53 and knockdown of p53 abolished oxLDL-induced activation of Bax and apoptosis. OxLDL activated p53 through production of mitochondria-derived reactive oxygen species. In EDCs treated with a recombinant adenovirus expressing superoxide dismutase or N-acetyl-cysteine (but not catalase), the p53-Bax pathway activated by oxLDL was blocked, and apoptosis was prevented. Of importance, treatment of EDC with low-concentration L5 stimulated superoxide dismutase expression, which significantly attenuated apoptosis in EDCs exposed to high-concentration L5. These findings suggest that exposure of EDCs and endothelial cells to either experimentally prepared or naturally occurring modified LDL results in an increased transfer of mitochondria-derived superoxide anion to p53, which stimulates a conformational change in Bax favoring its translocation to the mitochondria with resultant apoptosis of these cells.     

餐后血糖与血管并发症

越来越多的证据表明糖尿病、糖耐量异常和非糖尿病患者的餐后血糖异常与动脉硬化的发生发展关系密切,且较空腹血糖和糖化血红蛋白为强,在校正了其他心血管危险因素后,这种关系仍存在。其机制可能与餐后高血糖时脂质过氧化增强、内皮功能障碍、高凝状态、黏附因子水平升高、炎症、氧化/氮化应激增强等有关。阿卡波糖及其他药物的干预治疗能明显减少糖尿病、糖耐量异常的心血管事件。     

Advanced glycation end products and vascular inflammation: implications for accelerated atherosclerosis in diabetes

The formation of advanced glycation end products (AGEs) is an important biochemical abnormality that accompanies diabetes mellitus and, likely, inflammation in general. Here we summarize and discuss recent studies indicating that the effects of AGEs on vessel wall homeostasis may account for the rapidly progressive atherosclerosis associated with diabetes mellitus. Driven by hyperglycemia and oxidant stress, AGEs form to a greatly accelerated degree in diabetes. Within the vessel wall, collagen-linked AGEs may "trap" plasma proteins, quench nitric oxide (NO) activity and interact with specific receptors to modulate a large number of cellular properties. On plasma low density lipoproteins (LDL), AGEs initiate oxidative reactions that promote the formation of oxidized LDL. Interaction of AGEs with endothelial cells as well as with other cells accumulating within the atherosclerotic plaque, such as mononuclear phagocytes and smooth muscle cells (SMCs), provides a mechanism to augment vascular dysfunction. Specifically, the interaction of AGEs with vessel wall components increases vascular permeability, the expression of procoagulant activity and the generation of reactive oxygen species (ROS), resulting in increased endothelial expression of endothelial leukocyte adhesion molecules. AGEs potently modulate initiating steps in atherogenesis involving blood-vessel wall interactions, triggering an inflammatory-proliferative process and, furthermore, critically contribute to propagation of inflammation and vascular perturbation in established disease. Thus, a better understanding of the biochemical mechanisms by which AGEs contribute to such processes in the vessel wall could be relevant to devise preventive and therapeutic strategies for diabetic atherosclerosis.     

Glucose enhances human macrophage LOX-1 expression: role for LOX-1 in glucose-induced macrophage foam cell formation

Lectin-like oxidized LDL receptor-1 (LOX-1) is a newly identified receptor for oxidized LDL that is expressed by vascular cells. LOX-1 is upregulated in aortas of diabetic rats and thus may contribute to the pathogenesis of human diabetic atherosclerosis. In this study, we examined the regulation of human monocyte-derived macrophage (MDM) LOX-1 expression by high glucose and the role of LOX-1 in glucose-induced foam cell formation. Incubation of human MDMs with glucose (5.6 to 30 mmol/L) enhanced, in a dose- and time-dependent manner, LOX-1 gene and protein expression. Induction of LOX-1 gene expression by high glucose was abolished by antioxidants, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), nuclear factor-kappaB (NF-kappaB), and activated protein-1 (AP-1) inhibitors. In human MDMs cultured with high glucose, increased expression of PKCbeta2 and enhanced phosphorylation of extracellular signal-regulated protein kinase 1/2 was observed. Activation of these kinases was inhibited by the antioxidant N-acetyl-L-cysteine (NAC) and by the PKCbeta inhibitor LY379196. High glucose also enhanced the binding of nuclear proteins extracted from human MDMs to the NF-kappaB and AP-1 regulatory elements of the LOX-1 gene promoter. This effect was abrogated by NAC and PKC/MAPK inhibitors. Finally, high glucose induced human macrophage-derived foam cell formation through a LOX-1-dependent pathway. Overall, these results demonstrate that high glucose concentrations enhance LOX-1 expression in human MDMs and that this effect is associated with foam cell formation. Pilot data showing that MDMs of patients with type 2 diabetes overexpress LOX-1 support the relevance of this work to human diabetic atherosclerosis.