糖基化终末产物上调大鼠心脏微血管内皮细胞NF-kB和COX-2表达

目的研究糖基化终末产物（AGEs）对大鼠心脏微血管内皮细胞因子kB（NF-kB）和环氧合酶2（COX-2）表达的影响,探讨AGEs与糖尿病血管病变间的关系。方法体外培养大鼠心脏微血管内皮细胞至亚融和状态时,以不同浓度糖基化白蛋白BSA-AGEs与之作用不同时间后,检测内皮细胞NF-kB及COX-2蛋白的表达。结果25、50、100、200mg／LBSA-AGEs作用后,NF-kB和COX-2蛋白表达呈剂量依赖性增多,100mg／LAGEs作用6、12、24、48h,NF-kB和COX-2蛋白表达呈时间依赖性增多。结论BSA-AGEs可促进体外培养微血管内皮细胞NF-kB和COX-2的表达,其作用可能与NF-kB的活化有关。     

糖基化终末产物上调大鼠心脏微血管内皮细胞NF-κB和COX-2表达

目的 研究糖基化终末产物(AGEs)对大鼠心脏微血管内皮细胞因子κB(NF-κB)和环氧合酶2(COX-2)表达的影响,探讨AGEs与糖尿病血管病变间的关系. 方法 体外培养大鼠心脏微血管内皮细胞至亚融和状态时,以不同浓度糖基化白蛋白BSA-AGEs与之作用不同时间后,检测内皮细胞NF-κB及COX-2蛋白的表达. 结果 25、50、100、200mg/L BSA-AGEs作用后,NF-κB和COX-2蛋白表达呈剂量依赖性增多,100mg/L AGEs作用6、12、24、48h,NF-κB和COX-2蛋白表达呈时间依赖性增多. 结论 BSA- AGEs可促进体外培养微血管内皮细胞NF-κB和COX-2的表达,其作用可能与NF-κB的活化有关.     

糖基化终末产物对体外诱导大鼠视网膜微血管内皮细胞血管形成的影响

目的 应用Matrigel建立大鼠视网膜微血管内皮细胞血管形成的体外培养体系,研究糖基化终末产物对诱导视网膜微血管内皮细胞血管形成的影响,从而探讨糖基化终末产物在糖尿病微血管病血管新生中的作用.方法 体外培养视网膜微血管内皮细胞,制备糖基化白蛋白.在Matrigel上建立血管形成的体外培养体系,实验分浓度效应组和时间效应组,并进行CD34免疫细胞化学染色.采用光镜下计数管腔数及计算机图像分析对结果进行测定和分析.结果 经分离培养的视网膜微血管内皮细胞在Matrigel上培养形成管腔样结构.糖基化白蛋白(糖基化终末产物)作用于培养的视网膜微血管内皮细胞后,可见管腔形成数目增加,且在一定范围内呈时间和剂量依赖效应(P<0.01).结论 糖基化终末产物可以促进Matrigel体外诱导视网膜微血管内皮细胞的血管形成.     

糖基化终末产物诱导大鼠视网膜微血管内皮细胞表型和功能改变

目的 研究糖基化终末产物(AGEs)诱导大鼠视网膜微血管内皮细胞表型和功能的变化及相关机制.方法 SPF级雄性Wistar大鼠20只,7～8周龄,体重260～280 g,分离培养大鼠视网膜内皮细胞,采用免疫荧光染色、流式细胞术和体外形成毛细血管样网络结构的方法进行鉴定.视网膜内皮细胞在AGEs刺激后,用噻唑蓝(MTT)法分析细胞的增殖能力;用膜连蛋白V/碘化丙啶(Annexin V/PI)双染法检测细胞凋亡;采用逆转录聚合酶链反应(RT-PCR)和流式细胞术检测细胞AGEs受体、人蛋白激酶C(PKC)、细胞间黏附分子1(ICAM-1)和诱导型一氧化氮合酶(iNOS)的表达变化.采用t检验进行统计学分析.结果 分离纯化的大鼠视网膜内皮细胞表达血管性血友病因子(vWF)并在人工基膜上形成毛细血管网络结构.AGEs以时间和剂量依赖的方式抑制大鼠视网膜内皮细胞增殖能力,在200 mg/L的AGEs组培养至第5天时细胞增殖能力低于对照组(t=8.9,P＜0.05),7 d和9 d时抑制作用更明显(t值分别为15.7和46.1,均P＜0.01).400 mg/L AGEs组在第3天开始出现细胞增殖减慢(t=12.5,P＜0.05),从第5天开始增殖速度明显低于对照组(t值分别为22.4、41.5和77.7,均P＜0.01).并且AGEs诱导视网膜内皮细胞凋亡.进一步分析发现AGEs上调了大鼠视网膜内皮细胞AGEs受体、PKC、ICAM-1和iNOS的mRNA表达(t值分别为91.8、9.22、16和42,均P＜0.01)和蛋白水平的表达(t值分别为20.2、12.3、7.7和13.9,均P＜0.01).结论 AGEs可能通过上调AGEs受体诱导大鼠视网膜微血管内皮细胞表型和功能的改变.     

普罗布考对糖基化终末产物引发心脏微血管内皮功能紊乱的作用

目的:观察普罗布考（probucol）对糖基化终末产物（AGEs）作用后的心脏微血管内皮细胞iNOS表达的影响。方法: 原代培养心脏微血管内皮细胞,AGEs（100 mg/L）体外模拟高糖环境,在probucol(5 μmol/L,10 μmol/L,20 μmol/L) 作用后,检测ROS、NO和iNOS变化情况。结果: 与AGEs组比较,probucol组中ROS蛋白表达降低,NO生成增加,而 iNOS蛋白表达降低,且显示有浓度依赖性。结论: probucol可能通过对抗氧化应激的途径,缓解AGEs引发的心脏微血管内皮功能障碍。     

糖基化终末产物对视网膜微血管内皮细胞核因子κB活化的影响及辛伐他汀的保护作用

目的探讨糖基化终末产物对大鼠视网膜微血管内皮细胞核因子κB活化的影响及辛伐他汀的保护作用。方法体外培养大鼠视网膜微血管内皮细胞,制备糖基化终末产物—糖基化白蛋白。应用荧光显微镜观察核因子κB的活化。并观察给予辛伐他汀后视网膜微血管内皮细胞M atrigel上管腔形成的变化及单核细胞趋化蛋白1的表达。结果视网膜微血管内皮细胞无糖基化白蛋白刺激时,核因子κB主要表达在细胞浆;糖基化白蛋白刺激后核因子κB主要表达在核内,作用30 min时达高峰。糖基化白蛋白作用下管腔形成明显增多,单核细胞趋化蛋白1的表达明显增加,加入辛伐他汀后管腔形成明显减少,单核细胞趋化蛋白1的表达明显下降。结论糖尿病视网膜病变中糖基化终末产物发挥重要作用,核因子κB的活化是其关键环节;辛伐他汀可以减少糖基化白蛋白诱导的管腔形成及单核细胞趋化蛋白1的表达。     

形态学观察晚期糖基化终末产物诱导微血管内皮细胞核因子κB入核及其机制

目的 观察晚期糖基化终末产物(AGE)诱导的微血管内皮细胞核因子κB核转位,探讨氧化应激和内质网应激在核因子κB核转位中可能发挥的作用.方法 用AGE修饰的牛血清白蛋白(AGE-BSA)与人皮肤微血管内皮细胞(HDMEC)在体外共同培养1h,设立对照组进行比较,用免疫荧光化学染色示核因子κB的核转位情况;应用活性氧的抑制剂谷胱甘肽(GSH)、NADPH氧化酶(NOX)抑制剂Apocynin、内皮细胞高表达的NOX亚型NOX4的siRNA和内质网应激的标志性蛋白内质网转膜蛋白激酶1α(IRE1α)的siRNA分别预处理细胞后再给予AGE-BSA刺激,观察核因子κB的核转位情况.结果 与对照组相比,AGE-BSA可诱导人皮肤微血管内皮细胞核因子κB入核;应用GSH、Apocynin、NOX4siRNA和IRE1α siRNA预处理细胞均可抑制核因子κB的入核.结论 AGE对核因子κB的移位激活可能通过细胞内的氧化应激和内质网应激途径所介导.     

晚期糖基化终末产物致动脉粥样硬化的机制

晚期糖基化终末产物(advanced glycation end products,AGE)是由还原糖的羰基与游离氨基反应形成的复合物.AGE通过与其受体(RAGE)结合,改变细胞内信号转导、诱导炎症、增强氧化应激;与胶原交联、修饰脂蛋白等损害血管的完整性;这些导致血管内皮细胞功能紊乱,刺激平滑肌细胞迁移增殖,启动及加速糖尿病动脉粥样硬化和血管并发症的发生发展.阻断AGE-RAGE系统对防治糖尿病并发症具有重要意义.     

晚期糖基化终末产物致动脉粥样硬化的机制

晚期糖基化终末产物(advanced glycation end products,AGE)是由还原糖的羰基与游离氨基反应形成的复合物。AGE通过与其受体(RAGE)结合,改变细胞内信号转导、诱导炎症、增强氧化应激;与胶原交联、修饰脂蛋白等损害血管的完整性;这些导致血管内皮细胞功能紊乱,刺激平滑肌细胞迁移增殖,启动及加速糖尿病动脉粥样硬化和血管并发症的发生发展。阻断AGE-RAGE系统对防治糖尿病并发症具有重要意义。     

糖基化终末产物对人脐静脉内皮细胞凋亡及其受体表达的影响

目的 研究糖基化终末产物(AGEs)对人脐静脉内皮细胞(HUVEC)凋亡及其受体(RAGE)表达的影响。方法 采用流式细胞仪技术，荧光显微镜，电镜观察AGEs对培养的HUVEC凋亡的影响，同时用RT-PCR方法检测RAGE的mRNA的表达。结果 AGEs加入细胞培养中，可明显诱导HUVEC凋亡，并呈剂量依赖效应关系。在发生凋亡的同时有RAGE mRNA表达的增强。结论 AGEs对HUVEC有诱导凋亡的作用，该作用可能通过RAGE介导。     

AGEs对心肌微血管内皮细胞血管新生和管腔形成的影响

目的: 观察糖基化终产物（advanced glycation end products,AGEs）对心肌微血管内皮细胞(cardiac microvascular endothelial cells,CMECs)增殖能力、迁移能力,以及血管新生和管腔形成的作用和影响。方法: 以不同浓度（100、200和400 mg/L）的AGEs作用于CMECs 48 h,分别采用MTT比色法检测细胞的增殖能力;用Transwell法检测细胞的迁移能力;用毛细血管管腔结构形成试验检测AGEs对血管新生的影响。结果: CMECs在以不同浓度（100、200及400 mg/L）的AGEs作用48 h后,MTT比色法检测显示,吸光值（分别为0.1195±0.0049、0.1422±0.0058及0.1783±0.0220）明显高于对照组（0.0955±0.0161,P<0.05,P<0.01）;Transwell法检测细胞数［分别为(24.89±6.234)、(32.89±6.990)及(55.56±10.27)个］均高于对照组［（13.89±4.622）个,P<0.05,P<0.01］;管样结构的长度［分别为(3.261±0.7016)、(4.737±1.129)及(6.687±1.308)mm/mm2］均高于对照组［(2.089±0.6723)mm/mm2,P<0.05,P<0.01］。结论: AGEs可以促进CMECs血管新生和管腔结构的形成,且与其浓度呈正相关。     

非酶糖化蛋白对体外培养血管内皮细胞生长的影响

目的 观察糖代蛋白终末产物（ＡＧＥｓ）及高糖对体外培养人血管内皮细胞增殖的影响。方法 采用＾３Ｈ标记的腺嘧啶（＾３－ＴｄＲ）掺入法测定细胞增殖情况。结果 在含ＡＧＥｓ的培养液（５μｇ／ｍｌ）中增培养的新生儿脐带静脉内皮细胞,其２小时＾３Ｈ－ＴｄＲ掺入对照组的２．９５倍（Ｐ〈０．０１）,而在含３０ｍｍｏｌ／Ｌ葡萄糖的２液中培养的人胚胎脐带静脉内皮细胞,其２小时＾３Ｈ－ＴｄＲ掺入对照组（１１ｍｍｏｌ／     

Toxic action of advanced glycation end products on cultured retinal capillary pericytes and endothelial cells: relevance to diabetic retinopathy

Summary The toxic effects of advanced glycation end products (AGEs) on bovine retinal capillary pericytes (BRP) and endothelial cells (BREC) were studied. AGE-modified bovine serum albumin (AGE-BSA) was toxic to BRP. At a concentration of 500 μg/ml it reduced the BRP number to 48 ± 3 % (p < 0.05) of untreated controls, as determined by cell counting with haemocytometer. AGE-BSA was also toxic to bovine aortic endothelial cells (BAEC) reducing cell number to 84 ± 3.1 % of untreated controls. Under similar conditions, low concentrations (62.5 μg/ml) of AGE-BSA were mitogenic to BREC increasing the cell proliferation to 156 ± 11 % (p < 0.05) above that of untreated controls. At a higher dose of 500 μg/ml AGE-BSA decreased the proliferation of BREC to 85 ± 6 % of untreated controls. Immunoblot analysis demonstrated that BRP and BREC express the p60 AGE-receptor. Retinal capillary bed from the human also stained positively for the p60 AGE-receptor. Addition of 0.25 μg/ml of p60 AGE-receptor antibody was able to block the effects of AGE-BSA on BRP and BREC. The level of binding of [¹²⁵I]-labelled AGE-BSA to the cell surface was small but significant among the three cell types. There was also an increase in the internalized pool of radioligand in BRP and BREC but this was very much lower than in BAEC. In all the cell types the internalized pool of [¹²⁵I]-labelled AGE-BSA was much larger than the amount associated with the cell surface. Degradation products were not detected in the media over the 24-h incubation of the cells with [¹²⁵I]AGE-BSA. The binding of [¹²⁵I]-labelled AGE-BSA to the cell surface was prevented by the addition of p60 AGE-receptor. These results suggest that the interaction of AGE-modified proteins with the membrane-bound AGE-receptor may play an important role in the pathogenesis of diabetic retinopathy. [Diabetologia (1997) 40: 156–164] Received: 23 August 1996; and in revised form: 29 October 1996     

Association between serum levels of soluble receptor for advanced glycation end products and circulating advanced glycation end products in type 2 diabetes

Aims/hypothesis Activation of the receptor for advanced glycation end products (RAGE, also known as AGE-specific receptor [AGER]) has been implicated in the development of diabetic vascular complications. Blockade of RAGE using a soluble form of the receptor (sRAGE) suppressed vascular hyperpermeability and atherosclerosis in animal models. Since little is known about the regulation of endogenous sRAGE levels, we determined whether serum sRAGE is influenced by circulating AGEs and the severity of nephropathy in type 2 diabetic patients.Materials and methods We recruited 150 healthy control and 318 diabetic subjects. Diabetic subjects were subdivided into those with proteinuria, microalbuminuria or normoalbuminuria. Serum sRAGE was assayed by ELISA and serum AGEs by competitive ELISA using a polyclonal rabbit antiserum raised against AGE-RNase.Results Diabetic subjects had higher sRAGE (1,029.5 pg/ml [766.1–1,423.0] interquartile range vs 1,002.6 [726.5–1,345.3], p<0.05) and AGEs (4.07±1.13, SD, unit/ml vs 3.39±1.05, p<0.01) than controls. Proteinuric subjects had the highest sRAGE levels and there was a significant trend between the severity of nephropathy and sRAGE (p=0.01). In diabetic subjects, serum log(sRAGE) correlated with AGEs (r=0.27, p<0.001), log(plasma creatinine) (r=0.31, p<0.001), log(urine AER) (r=0.24, p<0.01) and log(triglycerides) (r=0.15, p<0.01). On stepwise linear regression analysis, AGEs and creatinine levels were the main independent determinants of sRAGE concentration.Conclusions/interpretation Serum sRAGE levels and circulating AGEs are associated with the severity of nephropathy in type 2 diabetic patients. Prospective studies are required to determine whether endogenous sRAGE potentially influences the development of diabetic vascular complications.     

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media

Aims/hypothesis: Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease. Since AGE-proteins have been hypothesized to mediate these effects, we studied the effects of AGE-bovine serum albumin on endothelial monolayer permeability and intracellular glutathione. Methods: AGE-BSA was prepared by incubating BSA for 30 days at 37 °C with 0.5 mol/l glucose and 0.2 mol/l phosphate buffer, pH 7.4. Permeability to fluorescently labelled BSA was assessed in a bovine pulmonary artery endothelial cell monolayer preparation. Glutathione was measured by an enzymatic assay. Results: AGE-BSA concentrations greater than 3 to 4 μmol/l produced maximal increases in permeability (6–8 times basal) within 3 to 4 h of incubation with the cells. This effect persisted for at least 48 h. However, BSA incubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeability-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at concentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (∼ 5 mmol/l), produced similar permeability increases at equivalent incubation times. Metal chelators (0.5 mmol/l) or inclusion of fetal bovine serum (10–20 %) blocked these permeability increases. These increases in permeability were associated with a decrease in endothelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a loss of cell-to-cell and cell-to-matrix adhesion molecules. Conclusion/interpretation: Trace amounts of redox-active metal ions in biological buffers could induce oxidative stress and alterations in cellular functions attributed to AGE-proteins in vitro. It is important to use metal-free phosphate and bicarbonate buffers in studies on cell biology in vitro, especially in serum-free media. [Diabetologia (2001) 44: 1310–1317] Received: 8 March 2001 and in revised form: 31 May 2001     

Role of advanced glycation end products in cardiovascular disease

Advanced glycation end products(AGEs) are produced through the non enzymatic glycation and oxidation of proteins,lipids and nucleic acids.Enhanced formation of AGEs occurs particularly in conditions associated with hyperglycaemia such as diabetes mellitus(DM).AGEs are believed to have a key role in the development and progression of cardiovascular disease in patients with DM through the modif ication of the structure,function and mechanical properties of tissues through crosslinking intracellular as well as extracellular matrix proteins and through modulating cellular processes through binding to cell surface receptors [receptor for AGEs(RAGE)].A number of studies have shown a correlation between serum AGE levels and the development and severity of heart failure(HF).Moreover,some studies have suggested that therapies targeted against AGEs may have therapeutic potential in patients with HF.The purpose of this review is to discuss the role of AGEs in cardiovascular disease and in particular in heart failure,focussing on both cellular mechanisms of action as well as highlighting how targeting AGEs may represent a novel therapeutic strategy in the treatment of HF.     

高级糖基化终末产物的合成及其与疾病的关系

高级糖基化终末产物(advanced glycation end products,AGEs)是由蛋白或者脂质暴露于还原糖中而形成的一组复杂且具有异质性的物质.该物质可通过内源性或外源性途径形成,大体可分为6种.AGEs可在不同种类的细胞内累积,影响细胞内及细胞外的结构和功能,同时它还可以通过和细胞表面的受体作用,通过信号传导,引发一系列的病理生理过程.AGEs沉积在细胞内,影响细胞功能,导致糖尿病血管并发症的发生.AGEs还与各种肿瘤的生物学特性相关,它可以修饰热休克蛋白27或者与AGEs受体相结合来影响肿瘤细胞的生长和浸润.AGEs的抑制物,如OPB-9195,可抑制这一系列病理生理过程.     

晚期糖基化终产物在糖尿病血管并发症中的作用机制

晚期糖基化终产物（AGEs）是蛋白质或脂质经过糖基化反应的产物。AGEs存在于糖尿病患者的血管中,促进动脉粥样硬化的发展。多种不同的细胞上AGEs的存在和聚集影响着细胞内外的结构和功能。AGEs通过与细胞外基质基底膜上的分子形成交联和与其受体（RAGE）结合引起多种微血管和大血管并发症。RAGE被AGEs激活后导致转录因子NF-κB和它的靶因子的表达。AGEs可诱发糜蛋白酶的表达和糜蛋白酶依赖性血管紧张素Ⅱ的生成,从而作用于糖尿病血管并发症的发展。