Effects Of Glycated/Oxidated Albumin On The Expression And Modulation Of Ezrin In Human Umbilical Vein Endothelial Cells

BackgroundPatients 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.Methods1. Preparation of AGE/AOPPAGE 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 RhoAfter 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 RhoAfter 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 ROCKAfter 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 ROCKAfter 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 Y27632HUVECs 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 oxidaseAfter 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 DPIAfter 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 ezrinAfter 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 ezrinAfter 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 SB203580After 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 rearrangementHUVECs 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 permeablityMonocyte 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. StatisticsAll 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.RESULTS1. 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 RhoRho 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 RhoRho 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 ROCKROCK 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 ROCKROCK 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 RAGEAGE-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 activation4. AGE-HSA or AOPP-HSA activating NADPH oxidase mainly through Rho/ROCK pathways(1) AGE-HSA activating NADPH oxidaseSerine 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 oxidaseSerine 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 pathwaysAGE-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 ezrinEzrin 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 ezrinEzrin 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 pathwayAGE-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 permeablityProtein 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).ConclusionsIn 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.