The Potential Role Of Ang-Tie2 Signaling In Regulation Of Endothelial Permeability

Objective:One of the major obstacles towards successful long-term peritoneal dialysis is the increased peritoneal transport rate after exposure to dialysis solutions. The mechanism of altered peritoneal transport is still not fully understood. AngiopoietinTie system is closely associated with vascular permeability, while its role in increased peritoneal transport rate is elusive. In the present study human umbilical vein endothelial cell line were cultured to investigate the effect of high concentration of glucose and Methylglyoxal(MGO, one of the glucose degeneration products in sterile dialysis solution) on endothelial permeability and the expression of junction molecules, as well as their effect on the expression of Ang2/Tie2 signaling. Furthermore, we evaluated whether COMP-Ang1(one of the engineered Ang1 variants) had impact on the permeability and junction molecules expression induced by high glucose and MGO.Methods: Human umbilical vein endothelial cell line(EA.hy926, passages 5-15) was grown in DMEM containing 10% fetal bovine serum at 37°C and 5% CO2. Endothelial cells(ECs) were exposed to 2.5% glucose, 2.5% mannitol and 200μM MGO respectively. The permeability of FITC-BSA was measuered to determine the endothelial permeability changes in each group, as well as the protein expression of Claudin-5, Occludin and VE-cadherin by western blotting. The m RNA and protein expression of Ang2/Tie2 signalling was tested by real-time PCR and western blot, respectively. Finally, ECs were cultured in 2.5% glucose, 2.5% mannitol, 200μM MGO with or without 150 ng/m L COMP-Ang1 to explore its role on ECs’ permeability and junction molecule expression.Result: 1. The incubation with 2.5% glucose(1.566±0.156 vs. 1, P=0.024), 2.5%mannitol(1.438±0.090 vs. 1, P=0.014) and 200μM MGO(1.482±0.135 vs. 1, P=0.003) resultedin an increased permeability of FITC-BSA in ECs, as well as downregulation of VEcadherin expression(for 2.5% glucose: 0.097±0.015 vs. 0.349±0.052, P=0.001; for 2.5% mannitol: 0.125±0.028 vs. 0.349±0.052, P=0.003; for 200μM MGO: 0.142±0.024 vs. 0.349±0.052, P=0.003), Occludin expression(for 2.5% glucose: 0.768±0.227 vs. 1.274±0.100,P=0.024; for 2.5% mannitol: 0.789±0.110 vs. 1.274±0.100,P= 0.005; for 200μM MGO: 0.826±0.110 vs. 1.274±0.100, P=0.006) and Claudin-5 expression(for 2.5% glucose: 0.171±0.028 vs. 0.313±0.047, P=0.011; for 2.5% mannitol: 0.195±0.034 vs. 0.313±0.047, P=0.025) in ECs. 2. The m RNA expression of Ang was significantly induced by 2.5% glucose(1.739±0.348 vs. 1, P=0.021) and 200μM MGO(1.389±0.239 vs. 1, P=0.048). The pretein expression of Ang was increased by 2.5% glucose(0.995±0.113 vs. 0.663±0.041, P=0.009), by 2.5% mannitol(0.900±0.129 vs. 0.663±0.041, P=0.038) and by 200μM MGO(0.886±0.084 vs. 0.663±0.041, P=0.015). MGO also stimulated the m RNA(1.231±0.059 vs. 1, P=0.048) and protein(0.737±0.090 vs. 0.54±0.114, P=0.023) expression of Tie2. 3. COMP-Ang1 as an Ang1 variant against Ang2/Tie2 signalling partly reduced the effect of glucose-induced increased permeability(1.235±0.119 vs. 1.566±0.156, P=0.027), and upregulated the protein exression of VE-cadherin(0.225±0.044 vs. 0.097±0.015, P=0.032), Claudin-5(0.605±0.038 vs. 0.467±0.044, P=0.008) and Occludin(1.133±0.069 vs. 0.768±0.227, P=0.024). COMP-Ang1 also reduced the effect of hypertonia-induced increased permeability(1.213±0.095 vs. 1.438±0.090, P=0.018), and upregulated the protein exression of VE-cadherin(0.125±0.028 vs. 0.313±0.054, P=0.006), Claudin-5(0.646±0.066 vs. 0.423±0.064, P=0.016) and Occludin(1.055±0.084 vs. 0.809±0.100, P=0.031). COMP-Ang1 also reduced the effect of MGO-induced increased permeability(1.216±0.079 vs. 1.482±0.135, P=0.009), and upregulated the protein exression of VE-cadherin(0.237±0.032 vs. 0.142±0.024, P=0.021), Claudin-5(0.751±0.051 vs. 0.561±0.100, P=0.046) and Occludin(1.056±0.078 vs. 0.826±0.110, P=0.042).Conclusions: Ang2/Tie2 contributed to the glucose-induced and MGO-induced increased permeability of ECs and to the downregulation of junction molecules expression. Ang2/Tie2 might be a potential treatment target for prevention of elevated vascular permeability in peritoneal membrane during long-term peritoneal dialysis.