摘要
研究背景
随着透析技术日臻成熟,慢性肾功能衰竭(chronic renal failure, CRF)病人因尿毒症致死者日益减少,目前导致这类病人死亡的主要原因是CRF和透析的各类并发症,心血管病是其中最重要的致死性并发症。CRF病人动脉粥样硬化的发生率是同年龄一般人群的10-20倍,且发病年龄提前至35-45岁,被称之为“加速性动脉粥样硬化”(accelerated atherosclerosis)。
CRF患者动脉粥样硬化快速发生、发展的原因目前尚未阐明,但一般推测与两方面因素有关：(1)CRF往往合并高血压、高脂血症、糖尿病等已被证实是一般人群中促发心血管病的危险因素(传统危险因素)；(2)CRF病人还合并一些与尿毒症有关的可能促发心血管病的因素,包括贫血、二价离子代谢紊乱、微炎症状态等(尿毒症相关的危险因素)。CRF时由于代谢紊乱,循环和组织中有多种糖和蛋白质代谢终产物潴留。晚期糖基化终产物(advanced glycation end products, AGE)和晚期蛋白氧化产物(advanced oxidation protein products, AOPP)是蛋白质被糖化/氧化修饰后的产物,在CRF患者体内异常升高,其水平与动脉粥样硬化的发病率呈正相关。本实验室近期用动物研究直接证实了AGE和AOPP修饰的蛋白对动脉粥样硬化的促进作用,国内外学者在进一步的体外实验研究中发现,AGE/AOPP修饰蛋白具有促发细胞炎症反应的作用。体内实验已证实AGE修饰的血清白蛋白,可显著促进动脉粥样斑块的形成,斑块部位血管内膜下的炎症细胞浸润显著增强。这些结果提示AGE/AOPP修饰蛋白可能参与了动脉粥样硬化发展过程中的急性炎症阶段,促进白细胞通过血管内皮屏障迁移至内膜下(transendothelial migration, TEM)。
TEM过程中,细胞骨架发生重组和再分布,形成应力纤维,最终导致细胞间隙增大,形成细胞旁通路；与此同时,内皮细胞膜表面的粘附分子重新分布,聚集在细胞表面的微突起、微绒毛等部位,引导活化的白细胞穿过细胞旁通路。而胞浆内细胞骨架重组和膜表面粘附分子重新分布均受埃兹蛋白、根蛋白、膜突蛋白(ezrin-radixin-moesin,ERM)蛋白家族的控制和协调。ERM蛋白一般处于非活化、非磷酸化状态,活化的ERM蛋白N端和粘附分子在胞内结合,C端和细胞骨架F-肌动蛋白结合,成为链接细胞表面粘附分子与胞浆内细胞骨架的“桥梁”。有研究表明,ERM蛋白同小GTP结合蛋白家族Rho/Rac信号通路的作用介导了细胞骨架和粘附分子的重新分布,提示ERM蛋白同Rho/Rac信号通路在TEM过程中发挥了重要的作用,但它们间的作用机制目前尚不清楚。
综上,AGE/AOPP可能参与了“加速性动脉粥样硬化”发展过程中的急性炎症阶段,促进白细胞通过血管内皮屏障迁移至内膜下。而ERM是介导TEM过程的关键分子,那么AGE/AOPP是否参与了对ERM蛋白活化的调控及其可能存在的机制,目前尚不清楚。本研究旨在观察AGE和AOPP修饰蛋白对人血管内皮细胞Ezrin活化的调控,并对上述作用的细胞内信号转导通路及分子机制进行探讨。
研究方法
一、AGE-HSA(human serum albumin)和AOPP-HSA的制备
1.体外制备无内毒素AGE-HSA
按文献方法体外制备无内毒素AGE-HSA。将1.75g/L纯化的HSA分别置于含或不含0.1mol/L D-葡萄糖的0.4M磷酸盐缓冲液中,37℃孵育8周;用pH7.4磷酸盐缓冲液透析以去除葡萄糖。以在同样条件但不含葡萄糖的缓冲液中孵育8周的HSA作为对照。用0.22μm的微孔滤膜过滤消毒。
2.体外制备无内毒素AOPP-HSA
将20mg/ml HSA与40mmol/l次氯酸等体积混合,室温放置30分钟,其摩尔比为1：140(HSA：次氯酸)。制备的AOPP-HSA在无内毒素PBS中透析24小时,除去游离的次氯酸。用0.22μm的微孔滤膜过滤除菌后4℃保存。20mg/ml HSA与PBS等体积混合作为对照。AOPP-HSA含量通过测定酸性条件下340nm的光吸收,以氯胺T为标准取得。
二、细胞培养
1、人脐静脉内皮细胞的分离与培养
分离培养人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cells, HUVECs):无菌条件下,取20-35cm新鲜人脐带(4℃保存不超过12小时),剪去两端各0.5cm,在脐静脉一端插入16号针头并固定,用温PBS灌洗3次至流出液清亮,用止血钳夹住脐带的另一端,灌入0.25%胰蛋白酶0.016%乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)消化液约8-15ml至脐静脉充盈,于37℃孵育7-9分钟,轻揉脐带,将消化液收集入50ml离心管,用PBS冲洗一遍,一并收入离心管中,离心沉淀(1000rpm)10分钟,弃上清,再用含15%胎牛血清(fetal bovine serum,FBS)的RPMI 1640培养液混匀细胞沉渣,调整细胞浓度至1.5-2.0×104/cm2种入培养瓶中,在37℃、5%CO2条件下培养。次日换液,除去未贴壁细胞,以后根据细胞生长情况每2-3日换液一次。用原代或第二代细胞进行实验。
2、THP-1细胞的复苏、培养
人THP-1单核细胞采用美国ATCC细胞株,复苏后在在37℃、5%CO2条件下含10%FBS的RPMI 1640培养液培养。实验前改换用无血清RPMI 1640培养液继续培养24小时,然后用于检测内皮细胞的通透性。
三、AGE/AOPP修饰白蛋白对Rho活性的影响
1.AGE-HSA对Rho活性的影响
观察AGE-HSA对Rho活性影响的剂量效应：HUVECs分别与25、50、100μg/ml AGE-HSA或100μg/ml未经修饰的HSA共同孵育5min,然后收集细胞总蛋白。观察AGE-HSA对Rho活性影响的时间效应：HUVECs与100μg/ml AGE-HSA或100μg/ml未经修饰的HSA共同孵育0、2、5、10及15min,然后收集细胞总蛋白。利用沉降法、Western Blotting检测Rho GTP,反映Rho的活性。阻断试验中,HUVECs与100μg/ml anti-RAGE预孵育1小时,用100μg/ml AGE-HSA刺激5min,然后收集细胞总蛋白。利用沉降法、Western Blotting检测Rho GTP,观察抗RAGE抗体对AGE-HSA所致Rho活性的影响。
2.AOPP-HSA对Rho活性的影响
观察AOPP-HSA对Rho活性影响的剂量效应：HUVECs与50、100、200μg/ml AOPP-HSA或200μg/ml未经修饰的HSA共同孵育5min,然后收集细胞总蛋白。观察AOPP-HSA对Rho活性影响的时间效应：HUVECs与200μg/mlAOPP-HSA或200μg/ml未经修饰的HSA共同孵育0、2、5、10及15min,然后收集细胞总蛋白。利用沉降法、Western Blotting检测]Rho GTP,反映Rho的活性。阻断试验中,HUVECs与100μg/ml anti-RAGE预孵育1小时,用200μg/ml AOPP-HSA刺激5min,然后收集细胞总蛋白。利用沉降法、Western Blotting检测Rho GTP,观察抗RAGE抗体对AOPP-HSA所致Rho活性的影响。
四、AGE/AOPP修饰白蛋白对Rho激酶(Rho kinase,ROCK)活性的影响
1.AGE-HSA对ROCK活性的影响
观察AGE-HSA对ROCK活性影响的剂量效应：HUVECs与25、50、100μg/ml AGE-HSA或100μg/ml HSA共同孵育10min,然后收集细胞总蛋白。观察AGE-HSA对ROCK活性影响的时间效应：HUVEC与100μg/ml AGE-HSA或相同浓度的HSA共同孵育0、5、10、15及30min,然后收集细胞总蛋白。Western Blotting检测肌球蛋白轻链(phosph-myosin light chain,p-MLC, ROCK的经典作用底物)的磷酸化水平表达,反映ROCK的活性。
2.AOPP-HSA对ROCK活性的影响
观察AOPP-HSA对ROCK活性影响的剂量效应：HUVECs与50、100、200μg/ml AOPP-HSA或200μg/ml HSA共同孵育10min,然后收集细胞总蛋白。观察AOPP-HSA对ROCK活性影响的时间效应：HUVECs与200μg/ml AOPP-HSA或相同浓度的HSA共同孵育0、5、10、15及30min,然后收集细胞总蛋白。Western Blotting检测MLC的磷酸化水平,反映ROCK的活性。
3.抑制剂对AGE-/AOPP-HSA所导ROCK活性的影响
在阻断试验中,细胞分别与anti-RAGE (100μg/ml)、ROCK抑制剂Y27632(30μM)预孵育1小时,用100μg/ml AGE-HSA或200μg/ml AOPP-HSA刺激10min,然后收集细胞总蛋白。Western Blotting检测MLC的磷酸化水平,观察抗RAGE抗体、Y27632对AGE-/AOPP-HSA所致ROCK活性的影响。
五、AGE/AOPP修饰白蛋白对NADPH氧化酶活性的影响1.AGE-HSA对NADPH氧化酶活性的影响
观察AGE-HSA对NADPH氧化酶活性影响的剂量效应：HUVECs与25、50、100μg/ml AGE-HSA或100μg/ml HSA共同孵育15min,然后收集细胞总蛋白。免疫沉淀、Western Blotting检测磷酸化-丝氨酸(phosph-serine,p-serine)、p47phox蛋白含量。
2.AOPP-HSA对NADPH氧化酶活性的影响
观察AOPP-HSA对NADPH氧化酶活性影响的剂量效应：HUVECs与50、100、200μg/ml AOPP-HSA或200μg/ml HSA共同孵育15min,然后收集细胞总蛋白。免疫沉淀、Western Blotting检测p-serine、p47phox蛋白含量。
3.抑制剂对AGE-/AOPP-HSA所致NADPH氧化酶活性的影响
在阻断试验中,细胞分别与anti-RAGE (100μg/ml)、Y27632(30μM) NADPH氧化酶抑制剂]DPI(100μM)预孵育1小时,用100μg/ml AGE-HSA或200μg/ml AOPP-HSA刺激15min,然后收集细胞总蛋白。免疫沉淀、Western Blotting检测p-serine、p47phox蛋白含量,观察抗RAGE抗体、Y27632、DPI对AGE-/AOPP-HSA所致NADPH氧化酶活性的影响。
六、AGE/AOPP修饰白蛋白对ezrin活性的影响
1.AGE-HSA对ezrin活性的影响
(1)观察AGE-HSA对ezrin活性影响的剂量效应：HUVECs与25、50、100μg/ml AGE-HSA或100μg/ml HSA共同孵育15min,然后收集细胞总蛋白。观察AGE-HSA对ezrin活性影响的时间效应：HUVECs与100μg/ml AGE-HSA或相同浓度的HSA共同孵育0、5、10、15及30min,然后收集细胞总蛋白。Western Blotting检测p-ezrin、ezrin蛋白含量。
(2)免疫荧光法观察p-ezrin在细胞内的分布。
2.AOPP-HSA对ezrin活性的影响
(1)观察AOPP-HSA对ezrin活性影响的剂量效应：HUVECs与50、100、200μg/ml AOPP-HSA或200μg/ml HSA共同孵育15min收集,然后细胞总蛋白。观察AOPP-HSA对ezrin影响的时间效应：HUVECs与200μg/ml AOPP-HSA或相同浓度的HSA共同孵育0、5、10、15及30min,然后收集细胞总蛋白。Western Blotting检测p-ezrin、ezrin蛋白含量。
(2)免疫荧光法观察p-ezrin在细胞内的分布。
3.抑制剂对AGE-/AOPP-HSA导致的ezrin活性的影响
在阻断试验中,细胞分别与anti-RAGE (100μg/ml)、Y27632 (30μM) DPI (100μM)、胞外信号调节激酶(extracellular signal-regulated kinase, ERK)抑制剂PD98059(10μM)及丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)P38抑制剂SB203580 (10μM)预孵育1小时,用100μg/ml AGE-HSA或200μg/ml AOPP-HSA,收集细胞总蛋白。Western Blotting检测p-ezrin、ezrin蛋白含量,观察抗RAGE抗体、Y27632、DPI、PD98059、SB203580对AGE-/AOPP-HSA所致ezrin活性的影响。
七、细胞骨架重构的观察、内皮细胞通透性的检测
1.细胞骨架重构的观察
HUVECs分别与100μg/ml AGE-HSA或相同浓度的HSA共同孵育8h,用罗丹明-鬼笔环肽染F-肌动蛋白(F-actin),免疫荧光显微镜观察应力纤维的形成。阻断试验中,HUVECs分别与anti-RAGE (100μg/ml)、Y27632 (30μM)、DPI(100μM)预孵育1小时,加入100μg/ml AGE-HSA共同孵育8h,用罗丹明-鬼笔环肽染F-肌动蛋白(F-actin),观察应力纤维的形成。
HUVECs分别与200μg/ml AOPP-HSA或相同浓度的HSA共同孵育8h,用罗丹明-鬼笔环肽染F-肌动蛋白(F-actin),免疫荧光显微镜观察应力纤维的形成。阻断试验中,HUVECs分别与anti-RAGE(100μg/ml)、Y27632(30μM)、DPI (100μM)预孵育1小时,加入200μg/ml AOPP-HSA共同孵育8h,用罗丹明-鬼笔环肽染F-肌动蛋白(F-actin),观察应力纤维的形成。
2.内皮细胞通透性的检测
Transewell模型检测内皮细胞的通透性,接种于inserts的HUVECs分别与100μg/ml AGE-HSA或相同浓度的HSA共同孵育8小时后,加入用无血清的RPMI1640悬浮的THP-1细胞,3小时后,收集下室的THP-1细胞,检测蛋白含量,反映穿过单层内皮细胞屏障的数目。阻断试验中,接种于小室的HUVECs分别与anti-RAGE (100μg/ml)、Y27632 (30μM)、DPI(100μM)预孵育1小时,加入100μg/ml AGE-HSA共同孵育8h,加入用无血清的RPMI 1640悬浮的THP-1细胞,3小时后,收集下室的THP-1细胞,检测蛋白含量,反映穿过单层内皮细胞屏障的数目。
接种于inserts的HUVECs分别与200μg/ml AOPP-HSA或相同浓度的HSA共同孵育8小时后,加入用无血清的RPMI 1640悬浮的THP-1细胞,3小时后,收集下室的THP-1细胞,检测蛋白含量,反映穿过单层内皮细胞屏障的数目。阻断试验中,接种于小室的HUVECs分别与anti-RAGE (100μg/ml)、Y27632 (30μM)、DPI (100μM)预孵育1小时,加入200μg/ml AOPP-HSA共同孵育8h,加入用无血清的RPMI 1640悬浮的THP-1细胞,3小时后,收集下室的THP-1细胞,检测蛋白含量,反映穿过单层内皮细胞屏障的数目。
八、统计方法
所有数据均代表3次重复实验的结果,以均数±标准差表示。所有统计由统计软件SPSS13.0完成。多个样本均数的比较采用One-Way ANOVA,两两比较采用LSD,P<0.05为差异有统计学意义。
结果
一、AGE-HSA和AOPP-HSA的鉴定
1.AGE-HSA的鉴定：
体外制备的修饰蛋白经荧光分光光度法鉴定,在激发波长365nm,吸收波长435nm测定制备的AGE-HSA中AGE的含量为123.194U/mg蛋白质,对照样本AGE含量为3.511U/mg蛋白质。所有制备的AGE-HSA或未经修饰的HSA经鲎试验法检测内毒素含量均低于0.25EU/ml。
2AOPP-HSA的鉴定：
制备的AOPP-HSA和未经修饰的HSA的蛋白浓度分别为9.4mg/ml和10.1mg/ml,AOPP含量分别为701μmol/l和1.55μmol/l,经蛋白浓度矫正后分别为72.7nmol/mg蛋白和0.14nmol/mg蛋白。所有制备的AOPP-HSA或未经修饰的HSA经鲎试验法检测内毒素含量均低于0.25EU/ml。
二、AGE-/AOPP-HSA通过RAGE活化Rho
1.AGE-HSA以剂量、时间依赖的方式活化Rho
HUVECs分别与25、50、100μg/ml的AGE-HSA共同孵育5min,Rho的活性随AGE-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与100μg/ml AGE-HSA共同孵育0、2、5、10及15min,Rho的活性随AGE-HSA刺激时间的延长而升高,在5min时达高峰,呈时间依赖性(P<0.01)。而未经修饰的HSA对Rho的活性无明显作用(P>0.05)。阻断试验中,AGE-HSA诱导的Rho活性增加可被anti-RAGE明显抑制(P<0.01)。
2.AOPP-HSA以剂量、时间依赖的方式活化Rho
HUVECs分别与50、100、200μg/ml的AOPP-HSA共同孵育5min,Rho的活性随AOPP-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与200μg/ml AOPP-HSA共同孵育0、2、5、10及15min,Rho的活性随AOPP-HSA刺激时间的延长而升高,在5min时达高峰,呈时间依赖性(P<0.01)。而未经修饰的HSA对Rho的活性无明显作用(P>0.05)。阻断试验中,AOPP-HSA诱导的Rho活性增加可被anti-RAGE明显抑制(P<0.01)。
三、AGE-/AOPP-HSA通过RAGE活化ROCK
1.AGE-HSA以剂量、时间依赖的方式活化ROCK
HUVECs分别与25、50、100μg/ml的AGE-HSA共同孵育10min,ROCK活性随AGE-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与100μg/ml AGE-HSA共同孵育0、5、10、15及30min,ROCK活性随AGE-HSA刺激时间的延长而升高,在10min时达高峰,呈时间依赖性(P<0.01)。而未经修饰的HSA对ROCK活性无明显作用(P>0.05)
2.AOPP-HSA以剂量、时间依赖的方式活化ROCK
HUVECs分别与50、100、200μg/ml的AOPP-HSA共同孵育10min,ROCK活性随AOPP-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与200μg/ml AOPP-HSA共同孵育0、5、10、15及30min,ROCK活性随AOPP-HSA刺激时间的延长而升高,在10min时达高峰,呈时间依赖性(P<0.01)。而未经修饰的HSA对ROCK活性无明显作用(P>0.05)
3.抑制剂抑制AGE-/AOPP-HSA诱导的ROCK活性增加
AGE-HSA或AOPP-HSA诱导的ROCK活性增加被anti-RAGE、Y27632明显抑制(P<0.01)。
四、AGE-/AOPP-HSA通过RAGE、Rho/ROCK活化NADPH氧化酶
1.AGE-HSA以剂量依赖的方式活化NADPH氧化酶
HUVECs分别与25、50、100μg/ml的AGE-HSA共同孵育15min,p-serine的表达量随AGE-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)；而未经修饰的HSA对NADPH氧化酶活化无明显作用(P>0.05)。
2.AOPP-HSA以剂量依赖的方式活化NADPH氧化酶
HUVECs分别与50、100、200μg/ml的AOPP-HSA共同孵育15min,p-serine的表达量随AOPP-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)；而未经修饰的HSA对NADPH氧化酶活化无明显作用(P>0.05)
3.抑制剂抑制AGE-/AOPP-HSA诱导的NADPH氧化酶活性的增加
AGE-/AOPP-HSA诱导的p-serine的表达量的上调被anti-RAGE、Y27632、DPI明显抑制(P<0.01)。
五、AGE-/AOPP-HSA通过RAGE、Rho/ROCK、NADPH氧化酶活化ezrin
1.AGE-HSA以剂量、时间依赖的方式活化ezrin
HUVECs分别与25、50、100μg/ml的AGE-HSA共同孵育15min,ezrin活性随AGE-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与100μg/ml AGE-HSA共同孵育0、5、10、15及30min,ezrin活性随AGE-HSA刺激时间的延长而升高,在15min时达高峰,呈时间依赖性(P<0.01)。而HSA对ezrin活性无明显作用(P>0.05)
免疫荧光化学染色可见HUVECs的胞膜和胞浆均有p-ezrin的表达,基础状态下有少量p-ezrin的表达,100μg/ml AGE-HSA作用15min,p-ezrin的表达明显增高。
2AOPP-HSA以剂量、时间依赖的方式活化ezrin
HUVECs分别与50、100、200μg/ml的AOPP-HSA共同孵育15min,ezrin活性随AOPP-HSA浓度的增加而升高,呈剂量依赖性(P<0.01)。HUVECs与200μg/ml AOPP-HSA共同孵育0、5、10、15及30min,ezrin活性随AOPP-HSA刺激时间的延长而升高,在15min时达高峰,呈时间依赖性(P<0.01)。而HSA对ezrin活性无明显作用(P＞0.05)
免疫荧光化学染色可见HUVECs的胞膜和胞浆均有p-ezrin的表达,基础状态下有少量p-ezrin的表达,200μg/ml AOPP-HSA作用15min,p-ezrin的表达明显增高。
3.抑制剂抑制AGE-/AOPP-HSA诱导ezrin的活化
AGE-HSA或AOPP-HSA诱导ezrin活性增加被anti-RAGE、Y27632、DPI明显抑制(P<0.01),而SB203580、PD98059未能抑制AGE-HSA或AOPP-HSA诱导的ezrin活性增加(P>0.05)
六、AGE-/AOPP-HSA诱导内皮细胞骨架重构、内皮细胞通透性增加
1.AGE-/AOPP-HSA诱导内皮细胞骨架重构
免疫荧光显微镜观察发现AGE-HSA和AOPP-HSA组应力纤维的形成量明显高于对照组,而anti-RAGE、Y27632、DPI能够减少AGE-HSA和AOPP-HSA诱导的应力纤维形成。同时,未经修饰的HSA对应力纤维形成无明显作用。
2.AGE-/AOPP-HSA诱导内皮细胞通透性增加
Transewell模型检测内皮细胞的通透性,收集下室的THP-1细胞,检测蛋白含量,结果发现AGE-HSA和AOPP-HSA组下室的蛋白浓度明显高于对照组,而anti-RAGE、Y27632、DPI能够抑制AGE-HSA和AOPP-HSA诱导致内皮细胞通透性增加的效应(P<0.01)。同时,未经修饰的HSA对内皮细胞通透性无明显作用(P>0.05)。
结论
我们的研究证实,AGE-/AOPP-HSA以时间和剂量依赖的方式增加ezrin蛋白的活化,其途径主要通过受体RAGE的介导依次活化了Rho/ROCK、NADPH氧化酶、ezrin,最终导致内皮细胞的骨架重构及细胞的通透性增加。
Background
Patients with chronic kidney disease (CKD) have a high burden of cardiovascular morbidity and mortality. The vast majority of CKD patients do have a significantly higher incidence of cardiovascular co-morbidities. The incidence of cardiovascular disease (CVD) in chronic renal failure (CRF) patients is 20- fold higher than that of non-CRF patients with similar ages, therefore, the CVD complication is also called "accelerated atherosclerosis".
The precise mechanism of atherosclerosis in patients with CRF is not yet known. Established risk factors, such as hypertension, dyslipidaemia and diabetes mellitus are involved in the pathogenesis of this phenomenon(traditional risk factors). Anemia, metabolic disorders of divalent ions, oxidative stress and micro-inflammatory state, represent emerging risk factors (uremia-related risk factors). Accumulation of advanced glycation end products (AGE)/advanced oxidation protein products (AOPP) has been found in patients with CKD. Serum levels of AGE/AOPP increase with the progression of renal failure and closely associated with occurrence of atherosclerosis. Our previous study demonstrated that intravenous infusion of AGE-/AOPP-modified albumin significantly increases macrophage infiltration in atherosclerotic plaques in hypercholesterolemic rabbits and in glomeruli in the remnant kidney model, suggesting that AGE/AOPP are not only the markers of oxidative stress, but potential inducers of vascular inflammation. AGE/AOPP could significantly increase the capacity of migration and the expressions of inflammatory mediators MCP-1, IL-6, VCAM-1. Recently we demonstrated that AGE/AOPP inhibits the production of nitric oxide (NO) but stimulates the production of MCP-1 by human endothelial cells (ECs) through activation of the p38 signal pathway in vitro. This effect may contribute to the pathogenesis of atherosclerosis. Consistent with the observation, accumulation of AGE/AOPP has been found in both experiments and human atherosclerotic lesions and has been linked to endothelial cells (ECs) dysfunction and monocyte activation. These data suggest that AGE/AOPP may participate in the process of "accelerated atherosclerosis" as inflammatory factors and promote white blood cells (WBC) to traverse the vascular endothelial barrier (transendothelial migration, TEM).
TEM is a critical step in the inflammatory response, which involves the spatiotemporal regulation of adhesion molecules, chemokines and cytoskeletal regulators. ECs cytoskeleton rearranged leads to the formation of stress fibers in the process of TEM. A number of molecules have been implicated in transmigration because genetic deletion or antibody blockade of these molecules impairs diapedesis. The ezrin/radixin/moesin(ERM) family of actin-binding proteins act both as linkers between the actin cytoskeleton and plasma membrane proteins and as signal transducers in responses involving cytoskeletal remodelling. Once ERM proteins are activated, they appear to be translocated from the cytosol to the plasma membrane, where they serve as membrane-cytoskeletal linkers. Both Rho GTPases and ERM proteins have been implicated in these responses. However, the mechanisms underlying ERM activation and function are so far unresolved.
As described above, both AGE/AOPP and the ERM proteins might be involved in TEM, then whether AGE/AOPP participates in the activation of ERM proteins is not yet clear. The present study was to evaluate the effects of glycated/oxidated albumin on the expression and modulation of ezrin in human umbilical vein endothelial cells. The signal transduction pathway that mediates the pathobiologic effects of AGE/AOPP was also explored in this study.
Methods
1. Preparation of AGE/AOPP
AGE was prepared in vitro according to the method described previous. Briefly, 1.75g/L purified HSA was incubated at 37℃for 8 weeks with or without 0.1mol/L D-glucose in 4 mM phosphate buffer. After incubation, all samples were dialyzed against phosphate-buffered solution, PH7.4. Samples incubated in the absence of glucose were used as controls.
AOPP was prepared in vitro as described previous. Briefly,20mg/ml fatty acid-free HSA was exposed to 40mmol/L HOCl for 30 min in the absence of free amino acid/carbohydrate/lipids to exclude formation of AGEs-like structures. The preparation was dialyzed overnight against PBS to remove free HOCl and passed through a DetoxiGel column to remove contaminated endotoxin. Endotoxin levels in the preparation were tested with limulus amebocyte lysate kit. AOPPs content was determined by measuring absorbance at 340 nm in acidic condition and was calibrated with Chloramines-T in the presence of potassium iodine.
2. Cell culture
(1)Human umbilical vein endothelial cells (HUVECs) were repared and cultured as follows:Human umbilical cords were collected immediately after delivery and stored in sterile containers at 4℃for a maximum period of 12 h. The veins were cannulated, washed with Phosphate Buffered Saline (PBS), and filled with Trypsin (37℃). After incubation in a waterbath (37℃for 7 min) the content of each vein was collected. The veins were washed once with PBS to harvest any remaining cells. Cells from each cord were centrifuged separately (1000×g for 10 min); the supernatant was discarded. Cells were cultured in RPMI 1640 supplemented with 15% fetal bovin serum (FBS) at 37℃,5%CO2. Only first-passage cells from one umbilicalord were used for one experiment.
(2)THP-1 cell culture Human THP-1 monocytes were obtained from ATCC. THP-1 were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum at 37℃,5%CO2. Prior to migration assays, monocytes were incubated overnight in serum-free medium for 24 hours.
3. Effect of AGE-/AOPP- modified HSA on the activation of Rho
(1) Effect of AGE-HSA on the activation of Rho
After cultured with serum-free medium for 12 hours, HUVECs were incubated with 25,50, 100μg/ml AGE-HSA for 5min or with 100μg/ml AGE-HSA for 0,2,5, 10 and 15min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Rho GTP was detected by pull down assay and western blotting. In some experiments, endotheilal cells were cultured with serum-free medium for 12 hours and pre-incubated with anti-RAGE for 1 hour, then 100μg/ml AGE-HSA were added and incubate with cells were for 5min. Rho GTP was detected as mentioned above.
(2) Effect of AOPP-HSA on the activation of Rho
After cultured with serum-free medium for 12 hours, HUVECs were incubated with50,100,200μg/ml AOPP-HSA for 5min or with 200μg/ml AOPP-HSA for 0,2, 5,10 and 15min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Rho GTP was detected by pull down assay and western blotting. In some experiments, HUVECs were cultured with serum-free medium for 12 hours and pre-incubated with anti-RAGE for 1 hour, then 200μg/ml AOPP-HSA were added and incubated with cells were for 5min. Rho GTP was detected as mentioned above.
4. Effect of AGE-/AOPP- modified HSA on the activation of ROCK
(1) Effect of AGE-HSA on the activation of ROCK
After cultured with serum-free medium for 12 hours, HUVECs were incubated with 25,50, 100μg/ml AGE-HSA for 10min or with 100μg/ml AGE-HSA for 0,5,10, 15 and 30min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Phosphorylation of MLC was detected by western blotting.
(2) Effect of AOPP-HSA on the activation of ROCK
After cultured with serum-free medium for 12 hours, HUVECs were incubated with50,100,200μg/ml AOPP-HSA for 10 min or with 200μg/ml AOPP-HSA for 0,5, 10,15 and 30min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Phosphorylation of MLC was detected by western blotting.
(3) Inhibitory effect of anti-RAGE and ROCK specific inhibitory Y27632
HUVECs were cultured with serum-free medium for 12 hours and pre-incubated with were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632 for one hour, 100μg/ml AGE-HSA or 200μg/ml AOPP-HSA were then added and incubate with cells for 10 minutes. Phosphorylation of MLC was detected as mentioned above.
5. Effect of AGE-/AOPP- modified HSA on the activation of NADPH oxidase
(1) Effect of AGE-HSA on the activation of NADPH oxidase
After cultured with serum-free medium for 12 hours, HUVECs were incubated with 25,50,100μg/ml AGE-HSA for 15min and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. P-serine and p47phox were detected by immunoprecipitation and western blotting.
(2) Effect of AOPP-HSA on the activation of NADPH oxidase
After cultured with serum-free medium for 12 hours, HUVECs were incubated with50,100,200μg/ml AOPP-HSA for 15 min and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. P-serine and p47phox were detected by immunoprecipitation and western blotting.
(3) Inhibitory effect of anti-RAGE、Y27632 and NADPH oxidase specific inhibitor DPI
After cultured with serum-free medium for 12 hours, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI for 1 hour, 100μg/ml AGE-HSA or 200μg/ml AOPP-HSA were then added and incubate with cells for 15min. P-serine and p47phox were detected as mentioned above.
6. Effect of AGE-/AOPP-modified HSA on the activation of ezrin
(1) Effect of AGE-HSA on the phosphorylation activity of ezrin
After cultured with serum-free medium for 12 hours, HUVECs were incubated with25,50,100μg/ml AGE-HSA for 15min or with 100μg/ml AGE-HSA for 0,5,10, 15 and 30min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Phosphorylation of ezrin was detected by western blotting. The expression and distribution of Ezrin in HUVECs was also detected by immunofluorescence.
(2) Effect of AOPP-HSA on the phosphorylation activity of ezrin
After cultured with serum-free medium for 12 hours, HUVECs were incubated with50,100,200μg/ml AOPP-HSA for 15min or with 200μg/ml AOPP-HSA for 0,5, 10,15 and 30min, respectively, and then were collected for protein isolation. Cells treated with medium alone or unmodified HSA were set as controls. Phosphorylation of ezrin was detected by western blotting. The expression and distribution of Ezrin in HUVECs was also detected by immunofluorescence.
(3) Inhibitory effect of anti-RAGE, Y27632, DPI, PD98059 and SB203580
After cultured with serum-free medium for 12 hours, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI, 10μM SB203580, 10μM PD98059 for 1 hour. 100μg/ml AGE-HSA or 200μg/ml AOPP-HSA were then added and incubate with cells for 15min.Phosphorylation activity of ezrin was detected as mentioned above.
7. Effects of AGE-/AOPP- modified HSA on EC cytoskeloton rearrangement and endothelial permeablity
(1) Effects of AGE-/AOPP-HSA on EC cytoskeloton rearrangement
HUVECs were incubated 100μg/ml AGE-HSA or the same concentration of HSA for 8 hours. The treated cells were incubated with rhodamine-phalloidin to stain F-actin to visualize the morphological changes of actin cytoskeleton. In some experiments, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI for 1 hour, then 100μg/ml AGE-HSA were added and incubate with cells for 8 hours. The morphological changes of actin cytoskeleton was detected as mentioned above.
HUVECs were incubated 200μg/ml AOPP-HSA or the same concentration of HSA for 8 hours. The treated cells were incubated with rhodamine-phalloidin to stain F-actin to visualize the morphological changes of actin cytoskeleton. In some experiments, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI for 1 hour, then 200μg/ml AOPP-HSA were added and incubate with cells for 8 hours.The morphological changes of actin cytoskeleton was detected as mentioned above.
(2) Effects of AGE-/AOPP-HSA on Endothelial monolayer permeablity
Monocyte diapedesis assays were performed with HUVECs cultured on cell culture inserts with an 8-mm pore size in24-well plates. After HUVECs were incubated 100μg/ml AGE-HSA or the same concentration of HSA for 8 hours, THP-1 cells resuspended in RPMI supplemented without serum were seeded into the upper chamber for 3 hours. Monocytes were collected from lower chambers collected for protein isolation. In some experiments, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI for 1 hour in upper chambers, then 100μg/ml AGE-HSA were added and incubate with cells for 8 hours. Monocyte diapedesis assays was detected as mentioned above.
After HUVECs were incubated 200μg/ml AOPP-HSA or the same concentration of HSA for 8 hours, THP-1 cells resuspended in RPMI supplemented without serum were seeded into the upper chamber for 3 hours. Monocytes were collected from lower chambers for protein isolation. In some experiments, HUVECs were pre-incubated with 100μg/ml anti-RAGE,30μM Y27632, 100μM DPI for 1 hour in upper chambers, then 200μg/ml AOPP-HSA were added and incubate with cells for 8 hours. Monocyte diapedesis assays was detected as mentioned above.
7. Statistics
All experiments were performed in triplicate. Continuous variables, expressed as mean±SEM, were compared using one-way ANOVA. Two-tailed P values,0.05 were considered statistically significant. Pairwise comparisons were evaluated by the Least-Significant-Difference procedure. Statistical analyses were conducted with SPSS 13.0 by Department of Biostatictics, Southern Medical University.
RESULTS
1. Characterization of AGE-/AOPP-HSA
The fluorescence spectra of the samples were measured at a protein concentration in a fluorescence spectrometer. Fluorescence excitation and emission maxima were 365 and 435 nm, respectively, which is characteristic of AGE proteins. AGE in the preparation of AGE and unmodified HSA were 123.194U/mg protein and 3.511U/mg protein respectively. The concentration of endotoxin in all preparations was lower than 0.25 EU/ml.
Protein concentrations of AOPP and unmodified HSA were 9.4mg/ml and 10.1mg/ml, respectively. The concentrations of AOPPs in samples were 701μmol/l and 1.55μmol/l, respectively. After calibrated the content of AOPPs were 72.7nmol/mg protein in prepared AOPPs-HSA and 0.14nmol/mg protein in unmodified HSA respectively. Endotoxin levels in the preparation were determined with the amebocyte lysate assay kit and were found to be below 0.25 EU/ml.
2. AGE-/AOPP-HSA activating Rho mainly through ligation of RAGE
(1) AGE-HSA activating Rho
Rho activation, as detected by both pull-down and western blotting was significantly increased by exposure of HUVECs with AGE-HSA but not HSA in a dose-and time-dependent manner. Rho activation was significantly suppressed by anti-RAGE, suggesting that RAGE played a central role in AGE-HSA induced Rho activation.
(2) AOPP-HSA activating Rho
Rho activation, as detected by both pull-down and western blotting was significantly increased by exposure of HUVECs with AOPP-HSA but not HSA in a dose-and time-dependent manner. Rho activation was significantly suppressed by anti-RAGE, suggesting that RAGE played a central role in AOPP-HSA induced Rho activation.
3. AGE-/AOPP-HSA activating ROCK mainly through ligation of RAGE
(1) AGE-HSA activating ROCK
ROCK activation, as detected by western blotting was significantly increased by exposure of HUVECs with AGE-HSA but not HSA in a dose-and time-dependent manner.
(2) AOPP-HSA activating ROCK
ROCK activation, as detected by western blotting was significantly increased by exposure of HUVECs with AOPP-HSA but not HSA in a dose-and time-dependent manner.
(3) AGE-HSA or AOPP-HSA activating ROCK mainly through ligation of RAGE
AGE-HSA or AOPP-HSA induced ROCK activation was significantly suppressed by anti-RAGE, Y27632, suggesting that RAGE played a central role in AGE-HSA or AOPP-HSA induced ROCK activation
4. AGE-HSA or AOPP-HSA activating NADPH oxidase mainly through Rho/ROCK pathways
(1) AGE-HSA activating NADPH oxidase
Serine phosphorylation, as detected by western blotting was significantly increased by exposure of HUVECs with AGE-HSA but not HSA in a dose-dependent manner.
(2) AOPP-HSA activating NADPH oxidase
Serine phosphorylation, as detected by western blotting was significantly increased by exposure of HUVECs with AOPP-HSA but not HSA in a dose-dependent manner.
(3) AGE-HSA or AOPP-HSA activating NADPH oxidase mainly through Rho /ROCK pathways
AGE-HSA- or AOPP-HSA-induced serine phosphorylation could be significantly blocked by pretreatment of HUVECs with anti-RAGE, Y27632, DPI, suggesting that Rho/ROCK pathways played a central role in AGE-HSA or AOPP-HSA induced NADPH oxidase activation.
5. AGE-/AOPP-HSA activating ezrin through RAGE-mediated signaling pathway
(1) AGE-HSA activating ezrin
Ezrin phosphorylation, as detected by both western blotting was significantly increased by exposure of HUVECs with AGE-HSA but not HSA in a dose-and time-dependent manner.
The expression of p-Ezrin protein was up-regulated by AGE-HSA but not HSA when detected with immunofluorescence and PI staining.
(2) AOPP-HSA activating ezrin
Ezrin phosphorylation, as detected by both western blotting was significantly increased by exposure of HUVECs with AOPP-HSA but not HSA in a dose-and time-dependent manner.
The expression of p-Ezrin protein was up-regulated by AOPP-HSA but not HSA when detected with immunofluorescence and PI staining.
(3) AGE-/AOPP-HSA activating ezrin through RAGE-mediated signaling pathway
AGE-HSA-or AOPP-HSA-induced ezrin phosphorylation could be significantly blocked by pretreatment of HUVECs with with anti-RAGE, Y27632, DPI, but not by SB203580, PD98059, suggesting interaction of AGE-HSA/AOPP-HSA with RAGE triggers an intracellular signaling pathway involving Rho GTPase、ROCK、NADPH oxidase and ezrin.
6. AGE-/AOPP-HSA leading to EC cytoskeloton rearrangement and increasing endothelial monolayer permeablity
(1) Morphology of F-actin in endothelial cells was changed greatly under the stimulation of AGE-/AOPP-HSA. Exposure of ECs to AGE-/AOPP-HSA caused a shift in F-actin distribution from web-like structure to polymerized stress fiber. And this change can be inhibited by pretreatment with anti-RAGE, Y27632 and DPI. The unmodified HSA did not affect morphology of actin cytoskeleton.
(2) Endothelial monolayer permeablity
Protein concentrations improved greatly under the stimulation of AGE-/ AOPP-HSA. And the changes can be inhibited by pretreatment with anti-RAGE, Y27632 and DPI (P<0.01). The unmodified HSA did not affect endothelial monolayer permeablity (P>0.05).
Conclusions
In the present study, we identified that the activation of vascular Ezrin protein by AGE-/AOPP-HSA increases gradually in a time- and dose-dependent manner through a RAGE-mediated signaling. Interaction of AGEs/AOPPs with RAGE triggers an intracellular signaling pathway involving Rho/ROCK、NADPH oxidase, leading to EC cytoskeloton rearrangement and increasing endothelial monolayer permeablity.
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