Effect Of Mdial On Advanced Glycation End Product-Induced Hyperpermeability In Endothelial Cells

Advanced glycation end products (AGEs) are a kind of generic term of relatively stable end products that are created through a nonenzymatic reaction between free amino groups of proteins and aldehyde groups of reducing sugars which forms Schiff base, following with Amadori reaction rearrangement. AGEs are closely associated with the development of diabetes and its complications, especially plays an important role in vascular permeability. Our previous work has confirmed that AGEs can cause contraction of endothelial cells, leading to increased permeability of endothelial cells and therefore increasing vascular permeability, by binding to its specific receptor RAGE and activating signal transduction mechanisms such as oxidative stress to influence the actin fabric and the structure and morphology of calcium-dependent adhesion protein,VE-cadherin,in endothelial cells.The Diaphanous-related formins family are defined as sub-class of formins family due to their specific functional domains, the GT-Pase binding domain GBD and Diaphanousauto-regulatory domain DAD, which are widely existed in a variety of eukaryotic cells from yeast to mammals. Previous studies have shown that mDial, one of the DRF family members, plays an important role in regulating cell adhesion, movement, cytokinesis, morphogenesis, cell polarity formation, serum response factor activation, and recent studies have implicated that the FH1 segment of mDial could combine with cytoplasmic segment of RAGE, and mDial is required in cell migration induced by RAGE ligand in transformed cell lines. Since our laboratory has confirmed that AGEs could affect the function and morphology of endothelial cells through binding to RAGE,, we persume that mDial is involved in HUVECs high-permeability triggered by AGEs.Objective:This study aims to elucidate whether mDial participates in hyperpermeability of HUVECs induced by AGEs, and explore the mechanisms.Methods:A series of experiments were accomplished in cell by using cell culture, western blot, fluorescent staining, flow cytometry, siRNA interference, transfected plasmids, adenovirus, and endothelial monolayer permeability assay, we observed levels of oxidative stress in HUVECs, the phosphorylation levels of tyrosine kinase Src, the phosphorylation levels of calcium-dependent adhesion protein VE-cadherin in endothelial cells and the changes of VE-cadherin distribution.AGE-modified bovine serum albumin (AGEs) was prepared by incubation of 150 mM bovine serum albumin with 250 mM of D-glucose for 8 weeks. Primary human umbilical vein endothelial cells were isolated, cultured in vitro and then seeded in 3.5cm,6cm,10cm culture dishes, micro small dish (petri dish), and the top of the chamber microporous membrane (6.5mm diameter,0.4?m pore size) in double transparent dish (transwell). Cells were grown to 90% confluent, and starved for 2h before treated respectively according to the protocals. We built the wild-type plasmid of RAGE or the mutant plasmids of RAGE which mutated the binding site of RAGE and mDial through changing the fifth arginine and the sixth glutamate that belongs to cytosolic segment of RAGE into alanine,to over express RAGE or down regulate RAGE and mDial interacation. Futher more, mDialsiRNA along with interference or overexpression adenovirus were selected to down or up regulate mDial expression. After the process of lipofectimine transfection and infection for 48h, the cells were incubated with AGEs 100mg/L,followed by the detection of the generation of reactive oxygen, phosphorylation level of Src and VE-cadherin, trans-endothelial electrical resistance, the permeability index of monolayer endothelial cells, and morphological changes of VE-cadherin.Results:1.mDial was associated with hyperpermeability of HUVECs induced by AGEs.(1). AGEs induced HUVECs hyperpermeability through AGE-RAGE binding.Transfection with RAGE siRNA for 48h before exposed to AGEs significantly increased TER and decreased hyperpermeability induced by AGEs, while control siRNA had no effect. The results implicated that AGEs induced HUVECs hyperpermeability through AGE-RAGE binding.(2). mDial was accosiated with the hyperpermeability of HUVECs induced by AGEs.Transfection with mDial siRNA, or infecting interference adenovirus, adenovirus of mDial, followed by AGEs, significantly reduced hyperpermeability induced by AGEs, while overexpression of mDial enhanced AGEs effect. These implicated that mDial was positively correlated with the hyperpermeability induced by AGEs.(3). mDial was involved in the process of hyperpermeability led by AGEs via binding to specific site of RAGE.Built the wild-type plasmid and the mutant plamids of RAGE which mutated the binding site of RAGE and mDial through changing the fifth arginine and the sixth glutamate that belongs to cytosolic segment of RAGE into alanine. Lipofectamine was used to transfer wild-type plasmid, mutant plasmid and control empty vector plasmid respectively into HUVECs, and 48 hours after infection,the cells were incubated with AGEs 100mg/L for another 8h.The results showed that RAGE mutant plasmid significantly increased TER, and reduced FITC-dextran leakage, while wild-type plasmid enhanced hyperpermeability induced by AGEs. These results implicated that mDial is involved in the process of hyperpermeability induced by AGEs via binding to specific site of RAGE.2.NADPH oxidase 4 (Nox4) was activated by AGEs to increase ROS level,which resulted in increased permeability of HUVECs, and mDialwas involved in this process via interaction with RAGE.(1). AGEs activated Nox4 to increase ROS level.HUVECs ROS level was detected by flow cytometry after stimulated by AGEs. The results showed that AGEs caused a time-dependent increase in ROS level. Compared with the only AGEs stimulation group, oxygen species ROS levels of Pre-transfection with Nox4 siRNA significantly reduced AGE-induced ROS. These data showed that AGEs evoked Nox4 activation to increase HUVECs ROS level.(2). mDial was involved in AGE-induced ROS production through interacting with RAGE.HUVECs were pre-transfected with mDialsiRNA or infected with interference adenovirus, overexpression adenovirus of mDial and corresponding control adenovirus for 48h, followed by 100mg/L AGEs for 15min. The results showed that mDial siRNA and interference adenovirus of mDial significantly reduced ROS production induced by AGEs, while overexpression adenovirus improved AGE-induced ROS production. Similarly the mutant plamids of RAGE significantly reduced ROS level, however wild type RAGE plasmid increased,AGE-induced ROS production. These data suggested that the interaction of RAGE and mDial mediated AGE-induced ROS production(3). mDial was involved in Nox4 membrane enrichment (translocation) in HUVECs induced by AGEs through interacting with RAGE.HUVECs were treated by AGEs, and then western blotting was used to detect the expression of Nox4 in membrane after extraction of membrane proteins. The results showed that the expression of Nox4 in the membrane of HUVECs reached a peak at 15min, this implicates that AGEs leads to Nox4 membrane enrichment in HUVECs, resulting in an increased level of oxidative stress. Pre-transfected with the mutant plamids of RAGE decreased Nox4 expression, while pre-transfected with the wild-type plasmids of RAGE was increased Nox4 membrane enrichment. These implicates that mDial was involved in Nox4 membrane enrichment in HUVECs induced by AGEs through interacting with RAGE.(4). Nox4 was involved in the process of increased permeability of HUVECs induced by AGEsTo confirm this role of Nox4, cells were transfected with Nox4 siRNA before AGEs stimulation. Knockdown of Nox4 significantly decreased AGE-induced endothelial hyperpermeability. These implicated that Nox4 was involved in the process of hyperpermeability induced by AGEs.3. AGEs caused phosphorylation of Src via interaction of RAGE and mDial.Cells were incubated with 100?g/mL of AGEs for different duration and phosphorylation of Src was determined by western blotting. We found that phosphorylation of Src Y419 was increased by AGEs in a time-dependent manner. This effect could be abolished by RAGE or mDialsiRNA, mutant plamids of RAGE or interference adenovirus of mDia1, while improved by wild-type plasmids of RAGE or overexpressed adenovirus of mDial. These results implicated that AGEs caused phosphorylation of Src via interaction of RAGE and mDial4. Oxidative stress was involved in increased phosphorylation of Src Y419 induced by AGEs.To further clarify the role of Oxidative stress in AGE-induced Src phosphorylation, HUVECs were pretreated with NADPH oxidase inhibitor Apocynin for 40 min or Nox4 siRNA for 48h, before stimulation with AGEs. We revealed that both Apocynin and Nox4 siRNA significantly attenuated AGE-induced Src phosphorylation. These implicated that oxidative stress was involved in increased phosphorylation of Src Y419 induced by AGEs, and that oxidative stress was on the upstream of Src.5. AGEs induced VE-cadherin Y658 phosphorylation required interaction of mDial and RAGE.We revealed that AGEs caused phosphorylation of VE-cadherin Y658 in a time-dependent manner. This effect could be inhibited by the mDial siRNA or mutant plamids of RAGE, while enhanced by wild-type plasmids of RAGE, which implicated that AGEs induced VE-cadherin Y658 phosphorylation required interaction of mDial and RAGE.6. mDial, RAGE and Nox4 were all involved in the dissociation of VE-cadherin conducted by AGEs.The morphological changes of VE-cadherin were observed by confocal laser microscope. We revealed that AGEs induced the dissociation of VE-cadherin, which was prevented by inhibition of RAGE-mDial-Nox4 signaling with RAGE, mDial or Nox4 siRNA, or interference adenovirus of mDial, but significantly promoted by overexpression adenovirus or wile-type plasmids of RAGE, These implicates that mDia1?RAGE?Nox4 were all involved in the morphological changes of VE-cadherin induced by AGEs.Conclusion:1. mDial is involved in AGEs-induced hyperpermeability in HUVECs by interacting with RAGE.2. mDial plays a key role in HUVECs oxidative stress caused by AGEs.3. AGEs lead to Src Y419 phosphorylation by activating mDial and oxidative stress.4. mDial participates in VE-cadherin Y658 phosphorylation triggered by AGEs.