Angiopoietin-like Protein 3 Modulates Human Glomerular Endothelial Cells Barrier Properties

BackgroundA 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. Partâ… The culture of the glomerular endothelial cell and establishment endothelial cell monolayerObjectives: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/cm².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/cm².Conclusions:Glomerular endothelial cell cultured in tissue-culture inserts can form a satisfactory monolayer.Partâ…¡Angiopoietin-like protein 3 modulates human glomerular endothelial cells barrier propertiesObjectives: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/cm²Â±4.52Ω/cm² vs 30.42Ω/cm²Â±1.45Ω/cm²,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/cm²Â±3.26Ω/cm² vs 30.61Ω/cm²Â±5.65Ω/cm²),19.5%(24.81Ω/cm²Â±2.53Ω/cm² vs 30.82±5.72Ω/cm²), 16.0%(25.76Ω/cm²Â±2.12Ω/cm² vs 30.6±5.76Ω/cm²) 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.Partâ…¢The mechanisms of angiopoietin-like protein 3 modulating human glomerular endothelial cells barrier propertiesObjectives: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 Ser⁴⁷³ 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.Conclusions1.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.