血管生成素样蛋白3调节人肾小球内皮细胞通透性的研究
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摘要
目前,越来越多的研究证实肾小球足细胞分泌的一些分子能影响肾小球内皮细胞的结构和功能,导致肾小球内皮细胞滤过功能的改变而参与蛋白尿的发生和发展,如血管内皮生长因子(vascular endothelial growth factor,VEGF)和血管生成素1(angiopoietin 1,Ang 1)。本课题组曾应用基因芯片技术发现微小病变肾病综合征患儿的血管生成素样蛋白3(angiopoietin-like protein 3,Angptl3)mRNA的表达水平显著高于正常儿童,及应用激光微切割技术分离肾组织发现在阿霉素大鼠肾病模型的肾小球内Angptl3 mRNA和蛋白的表达随着蛋白尿的加重而表达增高,并运用Western免疫印迹和实时荧光定量PCR技术发现体外培养的足细胞表达Angptl3,进一步还发现在微小病变和膜性肾病肾小球内Angptl3显著高于正常对照、薄基底膜肾病及局灶节段性肾小球硬化。以上实验结果显示Angptl3在肾小球内由足细胞分泌,并可能参与了蛋白尿的发生和发展。
血管生成素样蛋白是与血管生成素结构相似的糖蛋白,其含有N端的卷曲螺旋结构和C段的纤维蛋白原同源区域。研究发现Angptl3能与脐静脉内皮细胞整合素αvβ3纤维蛋白原同源区域结合,而其它研究已发现整合素αvβ3也是肾小球内皮细胞(glomerular endothelial cells,GEnCs)表达的整合素异构体之一。因此,我们推测在肾小球由足细胞分泌Angptl3可能通过与肾小球内皮细胞表面整合素αvβ3结合调节肾小球内皮细胞的通透性参与了蛋白尿的发生和发展。为此我们进行了以下三部分的研究,以探讨Angptl3是否能调节肾小球内皮细胞的通透性并研究其作用的可能机制。
第一部分肾小球内皮细胞培养和单层膜的建立
目的:观察肾小球内皮细胞在微孔细胞培养嵌套的滤膜上是否能形成生成致密连接的单层膜。
方法:以3-8代的GEnCs密度为1×10~5/cm~2接种于微孔细胞嵌套滤膜上。对滤膜肾小球内皮细胞行考马斯亮兰染色和定期测定滤膜肾小球内皮细胞电阻以确定GEnCs是否形成致密连接的单层膜。
结果:考马斯亮兰染色见GEnCs单层膜呈现为蓝色的片状,细胞间无间隙,表明GEnCs汇合成致密的单层。形成致密融合状态的肾小球内皮细胞单层膜电阻为(30.45±1.52)Ω/cm~2。
结论:在微孔细胞嵌套滤膜上生长的肾小球内皮细胞能形成致密的单层膜。
第二部分血管生成素样蛋白3调节肾小球内皮细胞通透性
目的:探讨血管生成素样蛋白3对肾小球内皮细胞通透性的影响。
方法:肾小球内皮细胞接种于微孔细胞培养嵌套的滤膜上,待细胞形成致密的单层膜后,分别给予不同浓度的Angptl3(0.02μg/ml、0.1μg/ml、0.5μg/ml、2.5μg/ml)作用1h;用浓度为0.5μg/ml Angptl3分别作用于GEnCs 1h、2h、3h。采用Millicell-ERS和两室弥散系统分别检测跨GEnCs单层膜电阻(TEER)和跨GEnCs异硫氰酸荧光素标记的白蛋白(FITC-BSA)滤过率。
结果:Angptl3可显著增加GEnCs的通透性,且呈剂量效应关系和时间效应关系。(1)Angptl3浓度达到0.1μg/ml始明显降低TEER和增加GEnCs对FITC-BSA的滤过率,分别为TEER降低33.2%(20.32Ω/cm~2±4.52Ω/cm~2 vs30.42Ω/cm~2±1.45Ω/cm~2)、FITC-BSA滤过率增加42.5%(0.17±0.045 vs0.12±0.019)(与对照组比较,P＜0.01),Angptl3浓度达到0.5μg/ml时,其对GEnCs的作用达到饱和;(2)0.5μg/ml Angptl3作用GEnCs1h、2h、3h可使TEER分别降低46.9%(16.19Ω/cm~2±3.26Ω/cm~2 vs 30.61Ω/cm~2±5.65Ω/cm~2)、19.5%(24.81Ω/cm~2±2.53Ω/cm~2 vs 30.82±5.72Ω/cm~2)、16.0%(25.76Ω/cm~2±2.12Ω/cm~2 vs30.6±5.76Ω/cm~2),可使FITC-BSA滤过率分别增加63.6%(0.20±0.032 vs0.12±0.027)、59.7%(0.1±0.056 vs 0.16±0.045)、50.9%(0.30±0.089 vs 0.2±0.072)(与对照组比较,P＜0.01)。
结论:血管生成素样蛋白3能导致肾小球内皮细胞通透性的增加,引起跨肾小球内皮细胞单层膜电阻下降和对FITC-BSA滤过率增加。
第三部分血管生成素样蛋白3调节肾小球内皮细胞通透性机制的研究
目的:探讨血管生成素样蛋白3调节肾小球内皮细胞的信号通路。
方法:(1)肾小球内皮细胞无血清饥饿1小时,再加入血管生成素样蛋白3(0.5μg/ml)或PBS作用1、2、3、4、5、6小时,Western blot检测不同处理时间点磷酸化Akt、总Akt、磷酸化FAK、总FAK、磷酸化ERK、总ERK的表达水平;(2)PI3K/Akt信号通路PI3K特异性抑制剂-LY294002(1μM)或MAPK信号通路的MEK特异性抑制剂-PD89095(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共作用1小时,测定肾小球内皮细胞单层膜电阻和对FITC-BSA的滤过率;(3)整合素αvβ3单克隆抑制剂-LM609(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共孵育1、3、5小时,Western blot检测磷酸化Akt和总Akt的表达水平;(4)LM609(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共孵育1小时,测定肾小球内皮细胞单层膜电阻和对FITC-BSA的滤过率。
结果:(1)Angptl3处理后肾小球内皮细胞磷酸化Akt和磷酸化FAK的表达水平显著增高(与对照组相比,P＜0.01),磷酸化的ERK无明显变化(与对照组比较,P＞0.05);(2)PI3K信号通路抑制剂LY294002预处理肾小球内皮细胞1小时能显著抑制Angpt13诱导的肾小球内皮细胞通透性的增高,能使Angpt13引起的跨肾小球内皮细胞的TEER下降减少约71%和对FITC-BSA滤过率的增加下降约76%(与血管生成素样蛋白3处理组比较,P＜0.01),而MAPK信号通路抑制剂PD98059对Angptl3诱导的肾小球内皮细胞的TEER的下降和对FITC-BSA滤过率的增加没有显著抑制作用(与血管生成素样蛋白3处理组比较,P＞0.05);(3)LM609(1μM)预处理肾小球内皮细胞1小时,能显著抑制Angptl3诱导的磷酸化Akt的增高(与血管生成素样蛋白3处理组比较,P＜0.01);(4)LM609(1μM)预处理肾小球内皮细胞1小时,能显著抑制Angptl3诱导的肾小球内皮细胞通透性的增高,能使Angptl3引起的跨GEnCs的TEER下降减少约74%和对FITC-BSA滤过率增加下降约77%(与血管生成素样蛋白3处理组比较,P＜0.01)。
结论:血管生成素样蛋白3可能通过整合素αvβ3、FAK/PI3K/Akt信号通路调节肾小球内皮细胞的通透性。
总结
1、血管生成素样蛋白3能显著增加肾小球内皮细胞的通透性,且呈剂量效应关系和时间效应关系;
2、血管生成素样蛋白3可能通过整合素αvβ3、FAK/PI3K/Akt信号通路调节肾小球内皮细胞的通透性。
Background
A number of studies have shown that some molecules produced by podocyte may influence glomerular endothelial function and contribute to the change of the glomerular filtration barrier,and some of them may take part in the development of proteinuria,such as vascular endothelial growth factor(VEGF),angiopoietin 1(angl). In our previous studies,by Affymetrix GeneChip technology we found the mRNA level of Angptl3 was significantly increased in kidneys of children with minimal change nephrotic syndrome compared to that of the normal control.And the mRNA and protein level of Angptl3 was increased in the glomerulus of adriamycin rats along with the development of proteinuria by laser microdissection.The level of Angptl3 in glomeruli of MCD and MN were significantly higher than those of normal control, TBMN or FSGS respectively.The expression of Angptl3 in cytoplasm of cultured podocyte was examined by reverse transcription and western blotting analyses.These preliminary data suggested that Angptl3 secreted by podocyte in glomeruli may regulate biological function of glomerular filtration barrier and may take part in development of proteinuria.
Angiopoietin-like proteins(Angptl) and angiopoietins are structurally resemblant glycoproteins characterized by two domains,a N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain.Experiments confirmed that Angptl3 could bind to the C-terminal fibrinogen homology domain of integrinαvβ3 and induce integrinαvβ3-dependent biological activity of umbilical venous endothelial cell,such as adhesion and migration.Integrinαvβ3 is also one of the main integrin heterodimers that GEnCs express.So,it was considered whether Angptl3 secreted by podocyte have effect on GEnCs via integrinαvβ3.The present work is part of our effort to understand the linkage between the Angptl3 secreted by podocyte and the biological functions of GEnCs.We investigate here whether Angptl3 could modulate human glomerular endothelial cell barrier properties via a possible signaling pathway.
PartⅠThe culture of the glomerular endothelial cell and establishment endothelial cell monolayer
Objectives:To investigate whether glomerular endothelial cell cultured in tissue-culture inserts form monolayer.
Methods:Polycarbonate supports in issue-culture inserts were seeded with GEnCs at passages 3 to 8 at 100,000 cells/cm~2.GEnCs monolayer were detected by coomassie brilliant blue staining and transendothelial resistance(TEER).
Results:Coomassie brilliant blue staining showed monolayer of GEnCs have no intercellular space.The TEER of a satisfactory monolayer were(30.45±1.52)Ω/cm~2.
Conclusions:Glomerular endothelial cell cultured in tissue-culture inserts can form a satisfactory monolayer.
PartⅡAngiopoietin-like protein 3 modulates human glomerular endothelial cells barrier properties
Objectives:To investigate the effect of angiopoietin-like protein 3 on the barrier properties of human glomerular endothelial cells.
Methods:The confluence GEnCs monolayer grown in tissue-culture inserts were cultured with ECM containing Angptl3 at various concentration(0.02μg/ml,0.1μg/ml,0.5μg/ml,2.5μg/ml) for 1h.GEnCs monolayer was treated with Angptl3 (0.5μg/ml) for 1,2,3h.Effect of Angptl3 on the permeability of GEnCs was assessed by transendothelial resistance(TEER) with Millicell-ERS system and by the diffusion of FITC-BSA across the GEnCs monolayer.
Results:Angptl3 increased significantly the permeability of GEnCs in a dose and time dependent way.(1)There was a marked increase in permeability of GEnCs after treatment with 0.1μg/ml Angptl3 for 1h,decreased by 33.2% (20.32Ω/cm~2±4.52Ω/cm~2 vs 30.42Ω/cm~2±1.45Ω/cm~2,P＜0.01) in TEER and increased by 42.5%(0.17±0.045 vs 0.12±0.019,P＜0.01 ) in permeability rates for FITC-BSA compared with control.When the concentration at 0.5μg/ml the effect of Angptl3 on GEnCs was maximum;(2)After treatment with 0.5μg/ml for 1,2,3h, TEER was decreased by 46.9%(16.19Ω/cm~2±3.26Ω/cm~2 vs 30.61Ω/cm~2±5.65Ω/cm~2),19.5%(24.81Ω/cm~2±2.53Ω/cm~2 vs 30.82±5.72Ω/cm~2), 16.0%(25.76Ω/cm~2±2.12Ω/cm~2 vs 30.6±5.76Ω/cm~2) respectively,and the permeability rates for FITC-BSA passing through GEnCs monolayer were increased by 63.6%(0.20±0.032 vs 0.12±0.027),59.7%(0.1±0.056 vs 0.16±0.045),50.9% (0.30±0.089 vs 0.2±0.072) respectively compared to that of control(P＜0.01).
Conclusions:These data suggest Angptl3 is able to increase human glomerular endothelial cell barrier properties.Angptl3 induce decrease in TEER and increase in permeability rates for FITC-BSA.
PartⅢThe mechanisms of angiopoietin-like protein 3 modulating human glomerular endothelial cells barrier properties
Objectives:To investigate the signaling pathway of angiopoietin-like protein 3 modulating human glomerular endothelil cells barrier.
Methods:(1)GEnCs were starved with serum-free medium for 1 h,and then GEnCs were treated with Angptl3(0.5μg/ml) or PBS for 1,2,3,4,5,6h.The level of phosphospecific Akt or total Akt,phosphospecific FAK,total FAK,phosphospecific ERK and total ERK were delected by Western blot.(2) The GEnCs monolayer was pretreated with LY294002(1μM),a PI3K inhibitor,or PD89095(1μM),a MEK inhibitor,for 1h,and treated with Angptl3(0.5μg/ml).TEER and FITC-BSA was measured at 1h.(3) GEnCs was pretreated with LM609(1μM),a integrinαvβ3 antibody,for 1h,and treated with Angptl3(0.5μg/ml) for 1h,3h,5h.The level of phosphospecific Akt or total Akt were delected by Western blot.(4) The GEnCs monolayer was pretreated with LM609(1μM) for 1h,and treated with Angptl3 (0.5μg/ml) for 1h.TEER and FITC-BSA was measured.
Results:(1) After GEnCs was treated with Angptl3(0.5μg/ml),the level of phosphospecific Akt and phosphospecific FAK at position Ser~(473) was markedly evaluated(P＜0.01),and the level of phosphospecific ERK didn't significantly change(P＞0.05).(2)LY294002(1μM) pretreatment blocked Angptl3-induced decrease in TEER and increase in passage of FITC-BSA of GEnCs.LY294002 prevented Angptl3-induced decrease in TEER by 71%and inhibited increase in FITC-BSA by 76%at 1h.PD98059(1μM) didn't inhibit Angptl3-induced decrease in TEER and increase in passage of FITC-BSA.(3) pretreatment of GEnCs with LM609 (1μM) could significantly blocked Angptl3-induced Akt phosphorylation.(4) pretreatment of GEnCs with LM609(1μM) inhibited Angptl3-induced decrease in TEER by in TEER by 74%and prevented increase in FITC-BSA by 77%.
Conclusions:These data suggested that Angptl3 could influence barrier properties of GEnCs via integrinαvβ3 and FAK/PI3K/Akt signaling pathway.
Conclusions
1.Angptl3 increased the permeability of GEnCs in a dose and time dependent way.
2.Angiopoietin-like protein 3 modulates human glomerular endothelial cells barrier properties via a possible signaling pathway involving in integrinαvβ3 and FAK/PI3K/Akt signaling pathway.
血管生成素样蛋白是与血管生成素结构相似的糖蛋白,其含有N端的卷曲螺旋结构和C段的纤维蛋白原同源区域。研究发现Angptl3能与脐静脉内皮细胞整合素αvβ3纤维蛋白原同源区域结合,而其它研究已发现整合素αvβ3也是肾小球内皮细胞(glomerular endothelial cells,GEnCs)表达的整合素异构体之一。因此,我们推测在肾小球由足细胞分泌Angptl3可能通过与肾小球内皮细胞表面整合素αvβ3结合调节肾小球内皮细胞的通透性参与了蛋白尿的发生和发展。为此我们进行了以下三部分的研究,以探讨Angptl3是否能调节肾小球内皮细胞的通透性并研究其作用的可能机制。
第一部分肾小球内皮细胞培养和单层膜的建立
目的:观察肾小球内皮细胞在微孔细胞培养嵌套的滤膜上是否能形成生成致密连接的单层膜。
方法:以3-8代的GEnCs密度为1×10~5/cm~2接种于微孔细胞嵌套滤膜上。对滤膜肾小球内皮细胞行考马斯亮兰染色和定期测定滤膜肾小球内皮细胞电阻以确定GEnCs是否形成致密连接的单层膜。
结果:考马斯亮兰染色见GEnCs单层膜呈现为蓝色的片状,细胞间无间隙,表明GEnCs汇合成致密的单层。形成致密融合状态的肾小球内皮细胞单层膜电阻为(30.45±1.52)Ω/cm~2。
结论:在微孔细胞嵌套滤膜上生长的肾小球内皮细胞能形成致密的单层膜。
第二部分血管生成素样蛋白3调节肾小球内皮细胞通透性
目的:探讨血管生成素样蛋白3对肾小球内皮细胞通透性的影响。
方法:肾小球内皮细胞接种于微孔细胞培养嵌套的滤膜上,待细胞形成致密的单层膜后,分别给予不同浓度的Angptl3(0.02μg/ml、0.1μg/ml、0.5μg/ml、2.5μg/ml)作用1h;用浓度为0.5μg/ml Angptl3分别作用于GEnCs 1h、2h、3h。采用Millicell-ERS和两室弥散系统分别检测跨GEnCs单层膜电阻(TEER)和跨GEnCs异硫氰酸荧光素标记的白蛋白(FITC-BSA)滤过率。
结果:Angptl3可显著增加GEnCs的通透性,且呈剂量效应关系和时间效应关系。(1)Angptl3浓度达到0.1μg/ml始明显降低TEER和增加GEnCs对FITC-BSA的滤过率,分别为TEER降低33.2%(20.32Ω/cm~2±4.52Ω/cm~2 vs30.42Ω/cm~2±1.45Ω/cm~2)、FITC-BSA滤过率增加42.5%(0.17±0.045 vs0.12±0.019)(与对照组比较,P＜0.01),Angptl3浓度达到0.5μg/ml时,其对GEnCs的作用达到饱和;(2)0.5μg/ml Angptl3作用GEnCs1h、2h、3h可使TEER分别降低46.9%(16.19Ω/cm~2±3.26Ω/cm~2 vs 30.61Ω/cm~2±5.65Ω/cm~2)、19.5%(24.81Ω/cm~2±2.53Ω/cm~2 vs 30.82±5.72Ω/cm~2)、16.0%(25.76Ω/cm~2±2.12Ω/cm~2 vs30.6±5.76Ω/cm~2),可使FITC-BSA滤过率分别增加63.6%(0.20±0.032 vs0.12±0.027)、59.7%(0.1±0.056 vs 0.16±0.045)、50.9%(0.30±0.089 vs 0.2±0.072)(与对照组比较,P＜0.01)。
结论:血管生成素样蛋白3能导致肾小球内皮细胞通透性的增加,引起跨肾小球内皮细胞单层膜电阻下降和对FITC-BSA滤过率增加。
第三部分血管生成素样蛋白3调节肾小球内皮细胞通透性机制的研究
目的:探讨血管生成素样蛋白3调节肾小球内皮细胞的信号通路。
方法:(1)肾小球内皮细胞无血清饥饿1小时,再加入血管生成素样蛋白3(0.5μg/ml)或PBS作用1、2、3、4、5、6小时,Western blot检测不同处理时间点磷酸化Akt、总Akt、磷酸化FAK、总FAK、磷酸化ERK、总ERK的表达水平;(2)PI3K/Akt信号通路PI3K特异性抑制剂-LY294002(1μM)或MAPK信号通路的MEK特异性抑制剂-PD89095(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共作用1小时,测定肾小球内皮细胞单层膜电阻和对FITC-BSA的滤过率;(3)整合素αvβ3单克隆抑制剂-LM609(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共孵育1、3、5小时,Western blot检测磷酸化Akt和总Akt的表达水平;(4)LM609(1μM)预处理肾小球内皮细胞1小时,再加入血管生成素样蛋白3(0.5μg/ml)共孵育1小时,测定肾小球内皮细胞单层膜电阻和对FITC-BSA的滤过率。
结果:(1)Angptl3处理后肾小球内皮细胞磷酸化Akt和磷酸化FAK的表达水平显著增高(与对照组相比,P＜0.01),磷酸化的ERK无明显变化(与对照组比较,P＞0.05);(2)PI3K信号通路抑制剂LY294002预处理肾小球内皮细胞1小时能显著抑制Angpt13诱导的肾小球内皮细胞通透性的增高,能使Angpt13引起的跨肾小球内皮细胞的TEER下降减少约71%和对FITC-BSA滤过率的增加下降约76%(与血管生成素样蛋白3处理组比较,P＜0.01),而MAPK信号通路抑制剂PD98059对Angptl3诱导的肾小球内皮细胞的TEER的下降和对FITC-BSA滤过率的增加没有显著抑制作用(与血管生成素样蛋白3处理组比较,P＞0.05);(3)LM609(1μM)预处理肾小球内皮细胞1小时,能显著抑制Angptl3诱导的磷酸化Akt的增高(与血管生成素样蛋白3处理组比较,P＜0.01);(4)LM609(1μM)预处理肾小球内皮细胞1小时,能显著抑制Angptl3诱导的肾小球内皮细胞通透性的增高,能使Angptl3引起的跨GEnCs的TEER下降减少约74%和对FITC-BSA滤过率增加下降约77%(与血管生成素样蛋白3处理组比较,P＜0.01)。
结论:血管生成素样蛋白3可能通过整合素αvβ3、FAK/PI3K/Akt信号通路调节肾小球内皮细胞的通透性。
总结
1、血管生成素样蛋白3能显著增加肾小球内皮细胞的通透性,且呈剂量效应关系和时间效应关系;
2、血管生成素样蛋白3可能通过整合素αvβ3、FAK/PI3K/Akt信号通路调节肾小球内皮细胞的通透性。
Background
A number of studies have shown that some molecules produced by podocyte may influence glomerular endothelial function and contribute to the change of the glomerular filtration barrier,and some of them may take part in the development of proteinuria,such as vascular endothelial growth factor(VEGF),angiopoietin 1(angl). In our previous studies,by Affymetrix GeneChip technology we found the mRNA level of Angptl3 was significantly increased in kidneys of children with minimal change nephrotic syndrome compared to that of the normal control.And the mRNA and protein level of Angptl3 was increased in the glomerulus of adriamycin rats along with the development of proteinuria by laser microdissection.The level of Angptl3 in glomeruli of MCD and MN were significantly higher than those of normal control, TBMN or FSGS respectively.The expression of Angptl3 in cytoplasm of cultured podocyte was examined by reverse transcription and western blotting analyses.These preliminary data suggested that Angptl3 secreted by podocyte in glomeruli may regulate biological function of glomerular filtration barrier and may take part in development of proteinuria.
Angiopoietin-like proteins(Angptl) and angiopoietins are structurally resemblant glycoproteins characterized by two domains,a N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain.Experiments confirmed that Angptl3 could bind to the C-terminal fibrinogen homology domain of integrinαvβ3 and induce integrinαvβ3-dependent biological activity of umbilical venous endothelial cell,such as adhesion and migration.Integrinαvβ3 is also one of the main integrin heterodimers that GEnCs express.So,it was considered whether Angptl3 secreted by podocyte have effect on GEnCs via integrinαvβ3.The present work is part of our effort to understand the linkage between the Angptl3 secreted by podocyte and the biological functions of GEnCs.We investigate here whether Angptl3 could modulate human glomerular endothelial cell barrier properties via a possible signaling pathway.
PartⅠThe culture of the glomerular endothelial cell and establishment endothelial cell monolayer
Objectives:To investigate whether glomerular endothelial cell cultured in tissue-culture inserts form monolayer.
Methods:Polycarbonate supports in issue-culture inserts were seeded with GEnCs at passages 3 to 8 at 100,000 cells/cm~2.GEnCs monolayer were detected by coomassie brilliant blue staining and transendothelial resistance(TEER).
Results:Coomassie brilliant blue staining showed monolayer of GEnCs have no intercellular space.The TEER of a satisfactory monolayer were(30.45±1.52)Ω/cm~2.
Conclusions:Glomerular endothelial cell cultured in tissue-culture inserts can form a satisfactory monolayer.
PartⅡAngiopoietin-like protein 3 modulates human glomerular endothelial cells barrier properties
Objectives:To investigate the effect of angiopoietin-like protein 3 on the barrier properties of human glomerular endothelial cells.
Methods:The confluence GEnCs monolayer grown in tissue-culture inserts were cultured with ECM containing Angptl3 at various concentration(0.02μg/ml,0.1μg/ml,0.5μg/ml,2.5μg/ml) for 1h.GEnCs monolayer was treated with Angptl3 (0.5μg/ml) for 1,2,3h.Effect of Angptl3 on the permeability of GEnCs was assessed by transendothelial resistance(TEER) with Millicell-ERS system and by the diffusion of FITC-BSA across the GEnCs monolayer.
Results:Angptl3 increased significantly the permeability of GEnCs in a dose and time dependent way.(1)There was a marked increase in permeability of GEnCs after treatment with 0.1μg/ml Angptl3 for 1h,decreased by 33.2% (20.32Ω/cm~2±4.52Ω/cm~2 vs 30.42Ω/cm~2±1.45Ω/cm~2,P＜0.01) in TEER and increased by 42.5%(0.17±0.045 vs 0.12±0.019,P＜0.01 ) in permeability rates for FITC-BSA compared with control.When the concentration at 0.5μg/ml the effect of Angptl3 on GEnCs was maximum;(2)After treatment with 0.5μg/ml for 1,2,3h, TEER was decreased by 46.9%(16.19Ω/cm~2±3.26Ω/cm~2 vs 30.61Ω/cm~2±5.65Ω/cm~2),19.5%(24.81Ω/cm~2±2.53Ω/cm~2 vs 30.82±5.72Ω/cm~2), 16.0%(25.76Ω/cm~2±2.12Ω/cm~2 vs 30.6±5.76Ω/cm~2) respectively,and the permeability rates for FITC-BSA passing through GEnCs monolayer were increased by 63.6%(0.20±0.032 vs 0.12±0.027),59.7%(0.1±0.056 vs 0.16±0.045),50.9% (0.30±0.089 vs 0.2±0.072) respectively compared to that of control(P＜0.01).
Conclusions:These data suggest Angptl3 is able to increase human glomerular endothelial cell barrier properties.Angptl3 induce decrease in TEER and increase in permeability rates for FITC-BSA.
PartⅢThe mechanisms of angiopoietin-like protein 3 modulating human glomerular endothelial cells barrier properties
Objectives:To investigate the signaling pathway of angiopoietin-like protein 3 modulating human glomerular endothelil cells barrier.
Methods:(1)GEnCs were starved with serum-free medium for 1 h,and then GEnCs were treated with Angptl3(0.5μg/ml) or PBS for 1,2,3,4,5,6h.The level of phosphospecific Akt or total Akt,phosphospecific FAK,total FAK,phosphospecific ERK and total ERK were delected by Western blot.(2) The GEnCs monolayer was pretreated with LY294002(1μM),a PI3K inhibitor,or PD89095(1μM),a MEK inhibitor,for 1h,and treated with Angptl3(0.5μg/ml).TEER and FITC-BSA was measured at 1h.(3) GEnCs was pretreated with LM609(1μM),a integrinαvβ3 antibody,for 1h,and treated with Angptl3(0.5μg/ml) for 1h,3h,5h.The level of phosphospecific Akt or total Akt were delected by Western blot.(4) The GEnCs monolayer was pretreated with LM609(1μM) for 1h,and treated with Angptl3 (0.5μg/ml) for 1h.TEER and FITC-BSA was measured.
Results:(1) After GEnCs was treated with Angptl3(0.5μg/ml),the level of phosphospecific Akt and phosphospecific FAK at position Ser~(473) was markedly evaluated(P＜0.01),and the level of phosphospecific ERK didn't significantly change(P＞0.05).(2)LY294002(1μM) pretreatment blocked Angptl3-induced decrease in TEER and increase in passage of FITC-BSA of GEnCs.LY294002 prevented Angptl3-induced decrease in TEER by 71%and inhibited increase in FITC-BSA by 76%at 1h.PD98059(1μM) didn't inhibit Angptl3-induced decrease in TEER and increase in passage of FITC-BSA.(3) pretreatment of GEnCs with LM609 (1μM) could significantly blocked Angptl3-induced Akt phosphorylation.(4) pretreatment of GEnCs with LM609(1μM) inhibited Angptl3-induced decrease in TEER by in TEER by 74%and prevented increase in FITC-BSA by 77%.
Conclusions:These data suggested that Angptl3 could influence barrier properties of GEnCs via integrinαvβ3 and FAK/PI3K/Akt signaling pathway.
Conclusions
1.Angptl3 increased the permeability of GEnCs in a dose and time dependent way.
2.Angiopoietin-like protein 3 modulates human glomerular endothelial cells barrier properties via a possible signaling pathway involving in integrinαvβ3 and FAK/PI3K/Akt signaling pathway.
引文
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[6] Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine[J]. Mol Cell, 2000, 6(4):909-919.
[7] Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family[J]. Cell Signal, 2003, 15(5):463-469.
[8] Ferrer I, Blanco R, Carmona M, et al. Phosphorylated mitogen-activated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activated protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies[J]. J Neural Transm,2001, 108(12):1397-1415.
[9] Arbabi S, Maier RV. Mitogen-activated protein kinases. Crit Care Med, 2002, 30(1 Supp):S74-S79.
[10] Hanks SK, Calalb MB, Harper MC, et al. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin[J]. Proc Natl Acad Sci USA, 1992, 89(18):8487-8491.
[11] Vacca A, Ria R, Presta M, et al. alpha(v)beta(3) integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells[J]. Exp Hematol, 2001, 29(8):993-1003.
[12]Han EK,Mcgonigal T,Wang J,et al.Functional analysis of focal adhesion kinase (FAK) reduction by small inhibitory RNAs[J].Anticancer Res,2004,24(6):3899-3905.
[13]Humphrey D,Rajfur Z,Vazquez ME,et al.In situ photoactivation of a caged phosphotyrosine peptide derived from focal adhesion kinase temporarily halts lamellar extension of single migrating tumor cells[J].J Biol Chem,2005,280(23):22091-22101.
[14]Schlaepfer DD,Mitra SK,Ilic D.Control of motile and invasive cell phenotypes by focal adhesion kinase[J].Biochim Biophys Acta,2004,1692(2-3):77-102.
[15]Tamura M,Gu J,Danen EH,et al.PTEN interactions with focal adhesion kinase and suppression of the extracellular matrix-dependent phosphatidylinositol 3-kinase/Akt cell survival pathway[J].J Biol Chem,1999,274(29):20693-20703.
1.Kramer-Zucker AG,Wiessner S,Jensen AM.Organization of the pronephric filtration apparatus in zebrafish requires Nephrin,Podocin and the FERM domain protein Mosaic eyes.Dev Biol,2005,285(2):316-329.
2.Nakhoul F,Ramadan R,Khankin E.Glomerular abundance of nephrin and podocin in experimental nephrotic syndrome:different effects of antiproteinuric therapies.Am J Physiol Renal Physiol.2005,289(4):F880-890.
3.Gerke P,Sellin L,Kretz O,et al.NEPH2(3)is located at the glomerular slit diaphragm,interacts with nephrin and is cleaved from podocytes by metalloproteinases.J Am Soc Nephrol,2005,16(6):1693-1702.
4.Liu G,Kaw B.Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability.J Clin Invest,2003,112(2):209-221.
5.Wolf G,Stahl RA.CD2-associated protein and glomerular disease.Lancet,2003,362(9397):1746-1748.
6.Huber TB,Schmidts M,Gerke P.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem,2003,278(15):13417-13421.
7.Xu ZG,Ryu DR,Yoo TH.P-Cadherin is decreased in diabetic glomeruli and in glucose-stimulated podocytes in vivo and in vitro studies.Nephrol Dial Transplant,2005,20(3):524-531.
8.Yaoita E,Kurihara H,Yoshida Y.Role of Fatl in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney.Kidney Int,2005,68(2):542-551.
9.Gerke P,Sellin L,Kretz O,et al.NEPH2 is located at the glomerular slit diaphragm, interacts with nephrin and is cleaved from podocytes by metalloproteinases. J Am Soc Nephrol, 2005, 16(6):1693-1702.
10.Ichimura K, Kurihara H, Sakai T. Actin filament organization of foot processes in rat podocytes. J Histochem Cytochem, 2003, 51(12): 1589-1600.
11.Patrie KM, Drescher AJ, Welihinda A. Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1. J Biol Chem, 2002, 277(33):30183-30190.
12.Vogtlander NP, Dijkman H, Bakker MA. Localization of alpha-dystroglycan on the podocyte: from top to toe. J Histochem Cytochem, 2005, 53(11):1345-1353.
13.Kretzler M. Regulation of adhesive interaction between podocytes and glomerular basement membrane. Microsc Res Tech, 2002, 57(4):247-253.
14.Eichler T, Ma Q, Kelly C. Single and combination toxic metal exposures induce apoptosis in cultured murine podocytes exclusively via the extrinsic caspase 8 pathway. Toxicol Sci, 2006, 90(2):392-9.
15.Susztak K, Raff AC, Schiffer M. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes, 2006, 55(1):225-233.
16.Yu D, Petermann A, Kunter U. Urinary podocyte loss is a more specific marker of ongoing glomerular damage than proteinuria. J Am Soc Nephrol, 2005 16(6):1733-1741.
17.Chen CA, Hwang JC, Guh JY. Reduced podocyte expression of alpha3betal integrins and podocyte depletion in patients with primary focal segmental glomerulosclerosis and chronic PAN-treated rats. J Lab Clin Med, 2006, 147(2):74-82.
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[1] Camenisch G, Pisabarro MT, Sherman D, et al. ANGPTL3 stimulates endothelial cell adhesion and migration via integrin alpha vbeta 3 and induces blood vessel formation in vivo[J]. J Biol Chem, 2002, 277(19):17281-17290.
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[5] Elmendorf JS, Pessin JE. Insulin signaling regulating the trafficking and plasma membrane fusion of GLUT4-containing intracellular vesicles[J]. Exp Cell Res, 1999, 253(1):55-62.
[6] Walker EH, Pacold ME, Perisic O, et al. Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine[J]. Mol Cell, 2000, 6(4):909-919.
[7] Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family[J]. Cell Signal, 2003, 15(5):463-469.
[8] Ferrer I, Blanco R, Carmona M, et al. Phosphorylated mitogen-activated protein kinase (MAPK/ERK-P), protein kinase of 38 kDa (p38-P), stress-activated protein kinase (SAPK/JNK-P), and calcium/calmodulin-dependent kinase II (CaM kinase II) are differentially expressed in tau deposits in neurons and glial cells in tauopathies[J]. J Neural Transm,2001, 108(12):1397-1415.
[9] Arbabi S, Maier RV. Mitogen-activated protein kinases. Crit Care Med, 2002, 30(1 Supp):S74-S79.
[10] Hanks SK, Calalb MB, Harper MC, et al. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin[J]. Proc Natl Acad Sci USA, 1992, 89(18):8487-8491.
[11] Vacca A, Ria R, Presta M, et al. alpha(v)beta(3) integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells[J]. Exp Hematol, 2001, 29(8):993-1003.
[12]Han EK,Mcgonigal T,Wang J,et al.Functional analysis of focal adhesion kinase (FAK) reduction by small inhibitory RNAs[J].Anticancer Res,2004,24(6):3899-3905.
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1.Kramer-Zucker AG,Wiessner S,Jensen AM.Organization of the pronephric filtration apparatus in zebrafish requires Nephrin,Podocin and the FERM domain protein Mosaic eyes.Dev Biol,2005,285(2):316-329.
2.Nakhoul F,Ramadan R,Khankin E.Glomerular abundance of nephrin and podocin in experimental nephrotic syndrome:different effects of antiproteinuric therapies.Am J Physiol Renal Physiol.2005,289(4):F880-890.
3.Gerke P,Sellin L,Kretz O,et al.NEPH2(3)is located at the glomerular slit diaphragm,interacts with nephrin and is cleaved from podocytes by metalloproteinases.J Am Soc Nephrol,2005,16(6):1693-1702.
4.Liu G,Kaw B.Neph1 and nephrin interaction in the slit diaphragm is an important determinant of glomerular permeability.J Clin Invest,2003,112(2):209-221.
5.Wolf G,Stahl RA.CD2-associated protein and glomerular disease.Lancet,2003,362(9397):1746-1748.
6.Huber TB,Schmidts M,Gerke P.The carboxyl terminus of Neph family members binds to the PDZ domain protein zonula occludens-1.J Biol Chem,2003,278(15):13417-13421.
7.Xu ZG,Ryu DR,Yoo TH.P-Cadherin is decreased in diabetic glomeruli and in glucose-stimulated podocytes in vivo and in vitro studies.Nephrol Dial Transplant,2005,20(3):524-531.
8.Yaoita E,Kurihara H,Yoshida Y.Role of Fatl in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney.Kidney Int,2005,68(2):542-551.
9.Gerke P,Sellin L,Kretz O,et al.NEPH2 is located at the glomerular slit diaphragm, interacts with nephrin and is cleaved from podocytes by metalloproteinases. J Am Soc Nephrol, 2005, 16(6):1693-1702.
10.Ichimura K, Kurihara H, Sakai T. Actin filament organization of foot processes in rat podocytes. J Histochem Cytochem, 2003, 51(12): 1589-1600.
11.Patrie KM, Drescher AJ, Welihinda A. Interaction of two actin-binding proteins, synaptopodin and alpha-actinin-4, with the tight junction protein MAGI-1. J Biol Chem, 2002, 277(33):30183-30190.
12.Vogtlander NP, Dijkman H, Bakker MA. Localization of alpha-dystroglycan on the podocyte: from top to toe. J Histochem Cytochem, 2005, 53(11):1345-1353.
13.Kretzler M. Regulation of adhesive interaction between podocytes and glomerular basement membrane. Microsc Res Tech, 2002, 57(4):247-253.
14.Eichler T, Ma Q, Kelly C. Single and combination toxic metal exposures induce apoptosis in cultured murine podocytes exclusively via the extrinsic caspase 8 pathway. Toxicol Sci, 2006, 90(2):392-9.
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