晚期糖基化终末产物通过氧化应激诱导大鼠软骨细胞损伤

目的:探讨晚期糖基化终末产物(advanced glycation end products,AGEs)能否通过氧化应激引起大鼠软骨细胞损伤。方法:原代培养SD大鼠软骨细胞,对细胞表型进行鉴定;应用CCK-8法检测软骨细胞生存率;DCFH-DA染色荧光显微镜下检测胞内活性氧簇(reactive oxygen species,ROS)的水平;Hoechst 33342核染色法及Annexin V-FITC/PI流式细胞法测定软骨细胞的凋亡率;RT-PCR法检测软骨细胞中Bax、Bcl-2、caspase-3、MMP3、MMP13和COL2的mRNA水平;Western blotting法检测软骨细胞中cleaved caspase-3、MMP3、MMP13和COL2的蛋白水平。结果:与对照组相比,AGEs可显著上调胞内ROS水平(P0.05),但经抗氧化剂N-乙酰半胱氨酸(NAC)抑制后ROS的生成明显减少(P0.05);另外,NAC可抑制AGEs引起的软骨细胞凋亡相关分子Bax/Bcl-2和caspase-3水平的上调,并减少MMP3和MMP13表达及COL2的丢失(P0.05)。结论:AGEs可通过氧化应激诱导大鼠软骨细胞损伤。     

晚期糖基化终末产物单克隆抗体制备与鉴定

目的：制备抗人晚期糖基化终末产物AGEs单克隆抗体（McAb）。方法：孵育D-葡萄糖和人血清白蛋白（HSA）获得AGE-HSA，取凝胶层析纯化后的第1峰AGE—HSA作为免疫原，免疫BALB／c小鼠，采用杂交瘤技术制备抗AGE．HSA的McAb，用间接ELISA和Western blot鉴定其亚类和特异性。结果：成功筛选出3株稳定分泌抗AGE-HSA的McAb杂交瘤细胞株，其分泌抗体亚类为IgM，经Western blot鉴定该McAb特异性高，亲和力强。结论：获得抗人AGE—HSA的McAb细胞株3株，为深入研究AGEs和应用提供了有力工具。     

梓醇抑制AGEs诱导的EA.hy926细胞炎症反应及RAGE表达

目的:研究梓醇对晚期糖基化终产物(AGEs)诱导的EA.hy926内皮细胞炎症反应的抑制作用并探讨其可能机制。方法:将常规培养的EA.hy926细胞随机分为对照组、梓醇对照组、AGEs组以及梓醇高剂量(0.5 mmol/L)、中剂量(0.25 mmol/L)和低剂量(0.05 mmol/L)保护组。激光共聚焦显微镜观察细胞内活性氧簇(ROS)的生成;RT-PCR和Western blot检测细胞中单核细胞趋化蛋白1(MCP-1)、肿瘤坏死因子α(TNF-α)、血管细胞黏附分子1(VCAM-1)及晚期糖基化终产物受体(RAGE)的mRNA及蛋白的表达。结果:梓醇保护组ROS生成均明显减少,MCP-1、TNF-α和VCAM-1的mRNA及蛋白表达均显著降低,RAGE蛋白表达明显受抑制,且呈剂量依赖性(P0.05)。结论:梓醇能够有效抑制AGEs诱导的EA.hy926细胞内氧化应激,减轻炎症反应,其机制可能与其降低RAGE表达有关。     

Dietary advanced glycation end products and their relevance for human health

Due to their bioactivity and harmful potential, advanced glycation end products (AGEs) are discussed to affect human health. AGEs are compounds formed endogenously in the human body andexogenously, especially, in foods while thermal processing. In contrast to endogenous AGEs, dietary AGEs are formed in much higher extent. However, their risk potential is also depending on absorption, distribution, metabolism and elimination. For over 10 years an intense debate on the risk of dietary AGEs on human health is going on. On the one hand, studies provided evidence that dietary AGEs contribute to clinical outcomes. On the other hand, human studies failed to observe any association. Because it was not possible to draw a final conclusion, the call for new interdisciplinary approaches arose. In this review, we will give an overview on the current state of scientific knowledge in this field. In particular, we focus on (I) the occurrence of AGEs in foods and the daily uptake of AGEs, (II) contribution to endogenous levels and (III) the effect on health-/disease-related biomarkers in humans.     

晚期糖基化终末产物对骨组织细胞代谢的影响

文题释义： 晚期糖基化终末产物：是还原糖(如葡萄糖)和某些代谢产物(如甲基已二醛)与蛋白质氨基经过非酶促反应生成的多种化合物,可以积聚在骨组织中,影响骨组织的结构和力学性能,导致骨强度显著下降。 骨胶原交联：骨胶原分子交联包括有利的酶催化交联(即未成熟的二价交联、成熟的三价交联)和不利的非酶催化交联。在骨组织发育过程中,胶原分子在酶催化的情况下,形成不成熟的二价交联,其中一部分二价交联进一步成熟形成三价交联;而在无酶催化情况下交联反应可形成晚期糖基化终末产物。 背景：随着骨组织工程学的研究和发展,发现晚期糖基化终末产物可以在骨组织中积累,影响骨骼的结构及生物力学性能。目前许多研究发现晚期糖基化终末产物/晚期糖基化终末产物受体通过特殊的作用机制后能够引起以成骨细胞、破骨细胞及骨细胞为主的骨组织细胞发生病理改变,导致骨重建失衡,骨骼强度下降,骨折发生率增加。 目的：综述晚期糖基化终末产物对骨骼生物力学的影响以及晚期糖基化终末产物/晚期糖基化终末产物受体对骨组织细胞的作用机制。 方法：由第一作者检索2005年1月至 2019年 7 月在PubMed、Web of Science 和 Medline数据库发表的有关晚期糖基化终末产物/晚期糖基化终末产物受体对骨组织细胞代谢的影响的文章,检索结果限于英文文献。 结果与结论：最终选取具有代表性的54篇文献进行归纳总结。晚期糖基化终末产物对骨胶原交联的影响,使得骨强度显著下降;晚期糖基化终末产物/晚期糖基化终末产物受体通过使骨组织细胞发生病理机制改变影响骨代谢,使得骨组织细胞发生本质改变。最终导致骨代谢平衡紊乱,骨骼脆性增加。骨质疏松症的发生与骨代谢相关的细胞活力改变有着直接关系,但具体相关作用机制需进一步研究,而这种特殊机制的改变在今后有可能为骨质疏松症提供独特的病理机制、诊断思维和相关治疗及预防策略。 中国组织工程研究杂志出版内容重点：人工关节;骨植入物;脊柱;骨折;内固定;数字化骨科;组织工程     

Understanding RAGE, the receptor for advanced glycation end products

Advanced glycation end products (AGEs), S100/calgranulins, HMGB1-proteins, amyloid- peptides, and the family of -sheet fibrils have been shown to contribute to a number of chronic diseases such as diabetes, amyloidoses, inflammatory conditions, and tumors by promoting cellular dysfunction via binding to cellular surface receptors. The receptor for AGEs (RAGE) is a multiligand receptor of the immunoglobulin superfamily of cell surface molecules acting as counter-receptor for these diverse molecules. Engagement of RAGE converts a brief pulse of cellular activation to sustained cellular dysfunction and tissue destruction. The involvement of RAGE in pathophysiologic processes has been demonstrated in murine models of chronic disease using either a receptor decoy such as soluble RAGE (sRAGE), RAGE neutralizing antibodies, or a dominant-negative form of the receptor. Studies with RAGE^–/– mice confirmed that RAGE contributes, at least in part, to the development of late diabetic complications, such as neuropathy and nephropathy, macrovascular disease, and chronic inflammation. Furthermore, deletion of RAGE provided protection from the lethal effects of septic shock caused by cecal ligation and puncture (CLP). In contrast, deletion of RAGE had no effect on the host response in delayed-type hypersensitivity (DTH). Despite the lack of effect seen in adaptive immunity by the deletion of RAGE, administration of the receptor decoy, sRAGE, still afforded a protective effect in RAGE^–/– mice. Thus, sRAGE is likely to sequester ligands, thereby preventing their interaction with other receptors in addition to RAGE. These data suggest that, just as RAGE is a multiligand receptor, its ligands are also likely to recognize several receptors in mediating their biologic effects.     

罗格列酮减轻糖基化终产物诱导的大鼠血管平滑肌细胞钙化

目的观察糖基化终产物(advanced glycation end products,AGEs)对血管平滑肌细胞钙化的影响以及罗格列酮对这种影响的干预作用,探讨罗格列酮在血管钙化产生中的作用。方法在含10mmol.L-1β-甘油磷酸培养液中加入200mg·mL-1的AGE-BSA及不同浓度(10-7mol.L-1～10-5mol.L-1)的罗格列酮体外培养大鼠血管平滑肌细胞,采用磷酸苯二钠法检测碱性磷酸酶活性,甲-酚酞络合酮方法测定细胞层钙含量,RT-PCR检测骨桥蛋白mRNA表达,Western blotting检测骨桥蛋白(osteopontin,OPN)蛋白表达。结果与对照组相比,AGE-BSA组细胞层钙含量增加、碱性磷酸酶活性升高,细胞OPNmRNA表达升高,OPN蛋白表达增加(P<0.01);10-7mol.L-1～10-5mol.L-1罗格列酮可抑制AGE-BSA对细胞的上述影响(P<0.01)。结论AGEs可促进血管平滑肌细胞钙化,罗格列酮可减轻AGEs对细胞的上述影响。     

Carnosine decreased oxidation and glycation products in serum and liver of high‐fat diet and low‐dose streptozotocin‐induced diabetic rats

High‐fat diet (HFD) and low‐dose streptozotocin (STZ)‐treated rats provide useful animal model for type II diabetes mellitus. Oxidative stress and advanced glycation end products (AGEs) play a role in the development of diabetic complications. Carnosine (CAR) has anti‐oxidant and anti‐glycating properties. We investigated the effects of CAR on oxidation and glycation products in HFD+STZ rats. Rats were fed with HFD (60% of total calories from fat) for 4 weeks, and then a single dose of STZ (40 mg/kg; i.p.) was applied. Rats with blood glucose levels above 200 mg/dl were fed with HFD until the end of the 12th week. CAR (250 mg/kg body weight; i.p.; five times a week) was administered to the rats for the last four weeks. CAR significantly decreased serum triglyceride (TG) (57.7%), cholesterol (35.6%) levels and hepatic marker enzyme activities of HFD+STZ rats. It significantly reduced serum reactive oxygen species (ROS) (23.7%), AGEs (13.4%) and advanced oxidized protein products (AOPP) (35.9%) and hepatic TG (59%), ROS (26%), malondialdehyde (MDA) (11.5%), protein carbonyl (PC) (19.2%) and AGE (20.2%) levels. Liver steatosis and hepatocyte ballooning were also significantly reduced. However, CAR treatment did not alter serum glucose and blood glycated haemoglobin and hepatic anti‐oxidant enzyme activities/mRNA expressions in HFD+STZ rats. Our results indicate that CAR decreased accumulation of oxidation and glycation products, such as MDA, AGE, AOPP and PC in the serum and liver and ameliorated hepatic dysfunction in HFD+STZ rats. This effect may be related to its anti‐oxidative, anti‐glycating, and anti‐lipogenic potential.     

糖尿病大鼠血管糖基化终产物含量与其受体的ICAM—1表达的关系

探讨糖尿病大鼠血管组织糖基化终产物（AGEs)含量与其受体（RAGE）和细胞间粘附因子－1（ICAM－1）表达的关系。复制糖尿病大鼠模型,采用荧光法、RT－PCR及原位杂交方法检测主动脉及心肌组织的AGEs含量以及RAGE和ICAM－1基因的表达。发现糖尿病大鼠主动脉和心肌组织AGEs含量升高（P＜0．01）;RAGE和ICAM－1基因表达增强（P＜0．05－0．05）;AGEs含量与RAGE及ICAM－1呈明显正相关（P＜0．01）;氨基胍治疗可缓解上述指标的变化。提示 AGEs可诱导RAGE和ICAM－1的表达。推测AGEs-RAGE相互作用是引起糖尿病血管内皮细胞功能紊乱和损伤的关键环节。     

Estimation of age of human cadavers by immunohistochemical assessment of advanced glycation end products in the hippocampus

AIMS: Advanced glycation end products (AGEs) are known to accumulate in long-lived tissue proteins during normal ageing. In this study, we examined the expression of AGEs in human hippocampus using immunohistochemistry and determined its utility for estimating the age of cadavers of unknown age. METHODS AND RESULTS: Hippocampus tissues were obtained at autopsy from 31 individuals, including 10 fire victims, aged 0--96 years within 3 days postmortem. Immunostaining using anti-AGE antibody demonstrated that the perikarya of pyramidal neurones in the hippocampus was immunoreactive for the anti-AGE antibody, and the immunoreactivity was increased with age. Quantitative analysis of the AGE-immunoreactivity in the pyramidal neurones of the CA4 region revealed a significant correlation between the AGE-immunoreactivity and the age in nonfire death cases with a correlation coefficient of 0.91 (P < 0.01). The significant correlation could be obtained even in fire death cases affected by the unusual environmental condition. CONCLUSIONS: These results suggest that the immunohistochemical analysis of AGEs in human hippocampus may be useful for the age estimation of cadavers with unknown age.     

糖基化终产物诱导心肌细胞炎症反应的研究

目的： 探讨糖基化终产物(AGEs)对乳鼠正常心肌细胞和胰岛素抵抗心肌细胞炎症反应的影响。方法: 原代培养乳鼠心肌细胞并建立胰岛素抵抗心肌细胞模型，用不同浓度葡萄糖孵育的糖化白蛋白（AGE-BSA）干预24 h，采用RT-PCR、免疫细胞化学染色和透射电镜法，观察AGE-BSA对细胞TNF-α mRNA和PPAR-γ mRNA表达、NF-κB活化以及细胞超微结构的影响。结果: AGE-BSA可诱导心肌细胞TNF-α mRNA表达和NF-κB活化,并可抑制PPAR-γ mRNA表达，与对照组及BSA组比较有显著差异（P<0.05）。BSA组与空白对照组比较差异无显著（P>0.05）。随着孵育葡萄糖浓度的增加，各AGE-BSA组间比较有显著差异（P<0.05）。AGE-BSA干预后胞内线粒体和滑面内质网增多、扩大。结论: AGEs能上调心肌细胞TNF-α mRNA表达和NF-κB活化，并可抑制PPAR-γ mRNA表达，提示AGEs在糖尿病心肌病的发生中可能有重要的作用。     

糖耐量异常与血管病变及糖基化终产物

糖耐量异常(impaired glucose tolerance,IGT)1979年由美国国家糖尿病资料组首先提出,按1999年WHO对IGT的诊断标准为:空腹血糖(fasting plasma glucose,FPG)小于7.0 mmol/L,OGTT后2 h血糖(2hPG)为7.8-11.1 mmol/L.近年,IGT患病率呈现显著增长的态势, IGT人群中每年以10 %-15 %的自然转归率发展为2型糖尿病(T2DM),是正常葡萄糖耐量(NGT)人群发生T2DM的100倍左右,并且IGT 具有可逆性.新诊断的IGT人群约有40%的患者存在血管病变,而糖基化终产物(advanced glycation end products, AGEs)与血管病变密切相关,IGT与AGEs也有一定关系,本文就三者之间的联系进行综述.     

25-hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid

Summary Samples of CSF and plasma were obtained simultaneously from 46 adult patients who had no endocrine disorders and were undergoing routine diagnostic lumbar puncture because of suspected or proved prolapse of a disc. Concentrations of 25-OHD, 24,25(OH)₂D and 1,25(OH)₂D were measured. The samples were purified by column chromatography and fractionated by HPLC. In the appropriate fractions the vitamin D metabolites were measured by PBA, and cytoreceptor assay. The results were as follows (median, range in brackets): 25-OHD in CSF 8.3 ng/ml (2.0–24.8), in plasma 14.5 ng/ml (7.0–36.0). 24,25(OH)₂D in CSF 1.8 ng/ml (0.3–4.6) and 2.5 ng/ml (0.4–4.7) in plasma. 1.25(OH)₂ D in CSF 25.0 pg/ml (2.2–39.0) and 31.0 pg/ml (10.1–55.0) in plasma. The correlations between plasma and CSF concentrations were as follows: 25-OHDr=0.479 (P<0.001); 24,25(OH)₂Dr=0.815 (P<0.001) and for 1.25(OH)₂Dr=0.497 (P<0.001).Our findings showed vitamin D metabolites to be present in human CSF.Abbreviations Ca Calcium - CSF Cerebrospinal fluid - Vitamin D₃ Cholecalciferol - CPM Counts per min - 24, 25 (OH)₂D 24, 25-dihydroxyvitamin D₃ - 1,25(OH)₂D 1,25-dihydroxyvitamin D₃ - Vitamin D₂ Ergocalciferol - HPLC High-pressure liquid chromatography - 25OHD 25-hydroxyvitamin D₃ - PTH Parathyroid hormone - PBA Protein binding assay - RIA Radioimmunoassay - D-CaBP Vitamin D dependent calcium-binding protein     

自噬对晚期糖基化终末产物致成纤维细胞损伤的早期保护

文题释义：细胞自噬：自噬是普遍存在于真核细胞中的一种高度保护的、维持细胞内环境自身稳定的细胞内降解机制。胞浆中的细胞器和蛋白质等首先被双层膜包裹,形成自噬体,随后自噬体与溶酶体融合形成自噬溶酶体,自噬可降解清除细胞内受损线粒体等细胞器及毒性蛋白质聚集体。细胞自噬可影响糖尿病等慢性创面成纤维细胞的活力,被认为是疾病修复的关键调节因素。晚期糖基化终末产物：是在持续高糖条件下,蛋白质、脂质甚至核酸的氨基与糖的醛基在非酶催化反应下生成的最终末产物。在糖尿病患者体内,病理性高血糖可加速糖基化反应,形成大量的晚期糖基化终末产物。局部晚期糖基化终末产物蓄积作为糖尿病代谢重构的直接产物,是皮肤组织细胞、细胞外基质和细胞因子改变的重要环境介质,使得糖尿病患者的皮肤易损伤并形成溃疡创面。背景：晚期糖基化终末产物与糖尿病创面难愈合密不可分,晚期糖基化终末产物可影响成纤维细胞增殖及迁移,但自噬在晚期糖基化终末产物致成纤维细胞损伤中的早期保护作用尚未见相关研究。目的：研究晚期糖基化终末产物对成纤维细胞活力及自噬的影响,探讨自噬在成纤维细胞损伤中的早期保护作用。方法：以体外传代培养的人成纤维细胞为研究对象,设立正常对照组(DMEM培养液)、晚期糖基化终末产物组(DMEM培养液+100 mg/L晚期糖基化终末产物)、晚期糖基化终末产物抑制剂N-苯乙酰噻唑组(DMEM培养   液+100 mg/L晚期糖基化终末产物+100 mg/L N-苯乙酰噻唑)、自噬抑制剂氯喹组(DMEM培养液+100 mg/L晚期糖基化终末产物+10 μmol/L氯喹)。培养48 h后,锥虫蓝染色检测各组细胞活力,Western blot法检测LC3及P62蛋白的表达,细胞免疫荧光实验检测自噬体形成,透射电镜观察自噬泡超微结构。结果与结论：①细胞死亡率：与正常对照组比较,晚期糖基化终末产物组细胞死亡率显著增加(P < 0.01);与晚期糖基化终末产物组比较,N-苯乙酰噻唑组细胞死亡率显著降低(P < 0.05),而氯喹组细胞死亡率显著增加(P < 0.05);②LC3及P62蛋白的表达：与正常对照组相比,晚期糖基化终末产物组LC3Ⅱ/LC3Ⅰ比值显著增加,P62表达显著降低(P < 0.01);与晚期糖基化终末产物组相比,N-苯乙酰噻唑组LC3Ⅱ/LC3Ⅰ比值明显下降,P62表达明显上升(P < 0.05),而氯喹组LC3Ⅱ/LC3Ⅰ蛋白含量变化不明显(P > 0.05),P62表达明显上升(P < 0.05);③自噬体形成：晚期糖基化终末产物组和氯喹组均可见细胞中LC3绿色荧光点状聚集物增加;晚期糖基化终末产物组可见数目较多的自噬小体和自噬泡结构,而氯喹组细胞中自噬体结构数量和空泡状结构少于晚期糖基化终末产物组;④结果证实,晚期糖基化终末产物可损伤成纤维细胞,降低细胞生存率,早期可激活细胞自噬;诱导的自噬对成纤维细胞损伤起到一定的保护作用。ORCID: 0000-0003-3966-8684(韩焱福)中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The antimicrobial peptide alpha defensin correlates to type 2 diabetes via the advanced glycation end products pathway

BackgroundDiabetes is a serious health problem that results in high mortality rates worldwide. α-defensins are antimicrobial peptides of the innate immune system that contribute to inflammation. However, data on serum levels of α-defensin in patients suffering from type 2 diabetes are limited.ObjectivesThis study aimed to assess the possible changes in α-defensin serum levels in patients suffering from type 2 diabetes and to investigate its correlation with relevant biomarkers.MethodologyAnalysis of serum α-defensin levels in 47 type 2 diabetics with diabetic neuropathy, 19 type 2 diabetics with no complications and 19 healthy control subjects by enzyme-linked immunosorbent assay was established. Furthermore, measurement of advanced glycation end products (AGEs) and fasting blood glucose (FBG) serum levels was performed, together with the lipid profile analysis.ResultsThe serum levels of α-defensin were higher in patients with and without diabetic neuropathy in comparison to control subjects. In addition, there was a significant correlation between α-defensin serum levels and AGEs and FBG serum levels as well as with the body mass index.Conclusionsα-defensins are significantly elevated in serum of type II diabetics, and correlate with AGEs serum levels indicating a crosstalk that may aggravate inflammation in type 2 diabetes.     

2型糖尿病大鼠骨折愈合障碍与体内晚期糖基化终末产物的变化

背景：近年来,晚期糖基化终末产物在骨组织领域的作用日益受到重视,而糖代谢紊乱是引起晚期糖基化终末产物增加的主要原因之一。 目的：观察2型糖尿病大鼠体内晚期糖基化终末产物表达的变化,并探讨其与糖尿病骨折愈合障碍的关系。 方法：30只SD大鼠随机均分为2组,实验组制备2型糖尿病模型,对照组正常饲养。糖尿病模型制备成功后,所有大鼠建立左胫骨骨折牵引成骨模型,胫骨延长0.3 mm/d,持续14 d。 结果与结论：牵引结束后,X射线摄片显示实验组糖尿病模型大鼠骨折断端之间牵引骨痂形成较对照组明显减少;骨痂组织学检查表现为微骨柱排列紊乱,初始基质前沿浅染。ELISA法检测实验组血清和双侧骨痂组织中晚期糖基化终末产物水平较对照组明显升高(P < 0.01),骨钙素明显降低(P < 0.01)。提示2型糖尿病大鼠骨折牵引骨痂生成障碍,而骨组织中晚期糖基化终末产物水平增高可能是导致2型糖尿病骨折愈合障碍的原因。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Immunofluorescence detection of advanced glycation end products (AGEs) in cookies and its correlation with acrylamide content and antioxidant activity

Food processing induces protein modifications by Maillard reactions. This generates advanced glycation end products (AGEs) that are known to affect human health. Therefore, it is of interest to monitor AGEs in food products. Currently Maillard products are detected by measuring fluorescence. However, several AGEs are non-fluorescent, while non-AGE components can exhibit autofluorescence. Therefore, specific AGE immunodetection was investigated. Immunofluorescence of AGEs as well as autofluorescence were determined in cookie extracts. Autofluorescence increases with baking time and sugar level, where AGE immunofluorescence increases with baking time until 20 minutes. Replacing sucrose by fructose confirmed the higher reactivity of fructose in AGE formation. The pattern of autofluorescence correlates well with the acrylamide and antioxidant activity. However, the immunodection of AGEs did not show such a correlation. At higher baking times the autofluorescence probably results from the generation of non-proteineious compounds. The immunofluorescence reduction likely results from the transient character of AGE epitopes.     

Advanced glycation end products induce actin rearrangement and subsequent hyperpermeability of endothelial cells

This study aimed to determine the effects of advanced glycation end products (AGEs) on endothelial cytoskeleton morphology and permeability, and to detect the underlying signaling mechanisms involved in these responses. Cultured endothelial cells (ECs) were exposed to AGE-modified human serum albumin (AGE-HSA), and EC cytoskeletal changes were evaluated by observing fluorescence of F-actin following ligation with labeled antibodies. Endothelial permeability was detected by measuring the flux of TRITC-albumin across the EC monolayers. To explore the signaling pathways behind AGE-induced EC alteration, ECs were treated with either soluble anti-AGE receptor (RAGE) IgG, or the MAPK inhibitors PD98059 and SB203580 before AGE-HSA administration. To further elucidate possible involvement of the ERK and p38 pathways in AGE-induced EC changes, adenovirus-carried recombinant constitutive dominant-negative forms of upstream ERK and p38 kinases, namely MEK1(A) and MKK6b(A), were pre-infected into ECs 24 h prior to AGE-HSA exposure. AGE-HSA induced actin cytoskeleton rearrangement, as well as EC hyperpermeability, in a dose and time-dependent manner. The effects were attenuated in cells pretreated with anti-RAGE IgG, PD98059 or SB203580, respectively. EC pre-infection with MEK1(A) and MKK6b(A) also alleviated the effect of AGEs. Furthermore, adenovirus-mediated administration of activated forms of either MEK1 or MKK6b alone induced rearrangement of F-actin and hyperpermeability. The results indicate that ERK and p38 MAPK play important roles in the mediation of AGE-induced EC barrier dysfunction associated with morphological changes of the F-actin.