Mechanism Of Advanced Glycation End Products-Induced Endoplasmic Reticulum Stress In Endothelial Cells

Advanced glycation end products (AGEs) are irreversibly crossed-linked protein derivatives formed by a non-enzymatic glycosylation reaction between aldehydes and the amino groups of proteins, followed by Schiff base and Amadori rearrangement. Accumulation of AGEs plays an important role in the endothelial injury durning the development of diabetic vascular complications. AGEs may alter protein structure or function via direct effects including formation of crosslinks that may trap neighboring unrelated macromolecules. Furthermore, AGEs and receptor for AGEs (RAGE) signaling exert complex effects on cellular functions via complicated transduction pathways, cytokine release or oxidative stress, which contributes to diabetic vascular complications.The endoplasmic reticulum (ER) in eukaryotic cells is a central organelle entrusted with protein folding and protein maturation, lipid biosynthesis, calcium store. Physiological states that increase the demand for protein folding, or stimuli that disrupt the reactions by which proteins fold, create an imbalance between the protein-folding load and the capacity of the ER, causing unfolded or misfolded proteins to accumulate in the ER lumen-a condition referred to as ER stress. To prevent such an accumulation of unfolded or misfolded proteins, eukaryotic cells have evolved the unfolded protein response (UPR), which alters a cell’s transcriptional and translational programs to cope with stressful conditions and to resolve the protein-folding defect. Prolonged stress leads to apoptosis and may thus be an important factor in the pathogenesis of cardiovascular diseases, especially obesity and type2diabetes mellitus.In resting cells, ER-stress sensors, including protein kinase-like ER kinase (PERK), inositol requiring la (IREla), and activating transcription factor6(ATF6), are maintained in an inactive state through association with the abundant ER chaperone-glucose regulated protein78(GRP78). In conditions of ER stress, GRP78is sequestered through binding to unfolded or misfolded proteins, thereby leading to the release and, consequently, the activation of the ER-stress sensors. Upon activation, these sensor molecules transduce a signaling cascade from ER to nucleus, and trigger the UPR.Objective:This study is to ascertain whether AGEs cause ER stress in human umbilical vein endothelial cells, and then to explore the cell signal transduction mechanism of AGEs-induced ER stress, especially the effects of oxidative stress in this process.Methods:Human umbilical vein endothelial cells were cultured and challenged with AGE-BSA. Western blot, siRNA and immunofluorescent staining were used to determine the expression of ER stress markers, the consequence of AGEs-induced ER stress and to explore the involvement of oxidative stress in cellular signal transduction mechanism of AGEs-induced ER stress.AGE-BSA was prepared by incubating bovine serum albumin (BSA) in PBS with D-glucose for8weeks in a sterile environment at37℃. HUVECs were seeded in35mm cell culture dishes, and serum-free medium was used to culture the cells for24h to obtain synchronous growth. Then the cells were treated respectively by the experiments requested. The expression of GRP78and the phosphorylation of IREla were detected by using western blot after the incubation of AGEs. The expression of IRE1α was down-regulated with IRE1α siRNA. The NF-κB nuclear translocation and activation was observed by immunofluorescent staining and western blot. HUVECs were incubated until the cells are60～80%confluent and then transfected with NADPH oxidase4(Nox4) siRNA for48h. Moreover, HUVECs were pretreated with15mmol/L reduced glutathione (GSH) for1h. Then the cells were cultured with100mg/L AGEs for12h. Next, the expression of GRP78and the phosphorylation of IRE1α were measured by western blot. The expression of Nox4was down-regulated with Nox4siRNA. The NF-kB nuclear translocation and activation was observed by immunofluorescent staining. Protein bands were visualized by chemiluminescence and then densitometric analysis was done by using Image J. TG was used as a positive control of ER stress in this study.Results:1. The expression and activity of ER-stress markers in HUVECs were increasesd by AGE-BSA(1) GRP78expression in HUVECs was remarkably increased by AGEs in time-and concentration-dependent manners.The results showed that GRP78expression was remarkably increased by AGE-BSA in time-(F=46.609, P=0.000) and concentration (F=10.265, P=0.000)-manners. In time-dependent experiments, the expression of GRP78increased gradually with the time extension. The significance occurred at12h (P=0.000), reached the peak at24h (P=0.000) and then began to decrease at48h (P=0.000) after AGE-BSA incubation. The treatment with increasing AGE-BSA concentration from12.5mg/L to200mg/L induced a gradual enhancement of GRP78expression in HUVECs, while the significance occurred at50mg/L AGE-BSA (P=0.016). Parallely, the expression of GRP78was also remarkably induced by treating HUVECs with positive ER stress inducer TG.(2) AGE-BSA induced the phosphorylation of IRE1α in time-and concentration-dependent manners.The results showed that the phosphorylation of IRE la was remarkably increased by AGE-BSA in time-(F=4.220, P=0.004) and concentration (F=5.739, P=0.000)-manners. In time-dependent experiments, the phosphorylation of IRE1α increased gradually with the time extension. The significance occurred at12h (P=0.025) and reached the peak at48h (P=0.004) after AGE-BSA incubation. The treatment with increasing AGE-BSA concentration from12.5mg/L to200mg/L induced a gradual enhancement of IRE1α phosphorylation in HUVECs, while the significance occurred at50mg/L AGE-BSA (P=0.031). Parallely, the phosphorylation of IREla was also remarkably induced by treating HUVECs with positive ER stress inducer TG.(3) AGE-BSA induced the phosphorylation of JNK in time-and concentration-dependent manners.The results showed that the phosphorylation of JNK was remarkably increased by AGE-BSA in time-(F=3.969, P=0.005) and concentration-(F=4.244, P=0.002) manners. In time-dependent experiments, the phosphorylation of JNK increased gradually with the time extension. The significance occurred at12h (P=0.026), reached the peak at24h (P=0.002) and then began to decrease at48h (P=0.005) after AGE-BSA incubation. The treatment with increasing AGE-BSA concentration from12.5mg/L to200mg/L induced a gradual enhancement of JNK phosphorylation in HUVECs, while the significance occurred at50mg/L AGE-BSA (P=0.011).2. The effect of ER stress inhibition on the activity of AGEs-induced NF-κB(1) NF-κB activity was increased by AGEs treatmentHUVECs were exposed to100mg/L AGE-BSA for12h and the nuclear extracts were collected. NF-κB contained in the nuclear extracts was detected by western blotting. The results showed that compared to control group, the amount of NF-κB in nucleus was significantly increased by AGE-BSA treatment (P=0.000). It suggested that AGE-BSA application induced the activation of NF-κB through enhancement of NF-nuclear translocation.(2) The effect of ER stress inhibition on the activity of NF-κB induced by AGEsHUVECs were incubated until the cells are60～80%confluent and then transfect with IRE1α siRNA and control siRNA respectively for48h. Total proteins were collected and the knockdown of the target molecule, IRE1α, was monitored by western blotting. The results showed that the expression of IRE1α protein was decreased by IRE1α siRNA compared to control group but there was no difference between control siRNA and control group. The results suggested that the expression of IRE1α were down-regulated by IRE1α siRNA.HUVECs were transfected with IRE la siRNA and control siRNA respectively, and then stimulated with100mg/L AGE-BSA for12h. Nuclear extracts were collected and nuclear NF-κB p65was determined by western blotting. The results showed that in HUVECs transfected with IRE la siRNA, the amount of nuclear NF-κB p65did not increase after AGE-BSA treatment, while control siRNA showed no effects on AGE-BSA-induced increase of nuclear NF-κB p65. The results suggested that inhibition of ER stress attenuated the activation of NF-κB induced by AGEs.3. The effect of ER stress inhibition on the nuclear translocation of AGEs-induced NF-κB by morphology(1) NF-κB nuclear translocation induced by AGEsHUVECs were incubated with100mg/L AGE-BSA and NF-κB localization was viewed in confocal laser scanning microscope by immunofluorescent staining. The results displayed the translocation of NF-κB from the cytoplasm to the nucleus upon the stimulation of AGE. Morphologically, the results confirm that AGE-BSA induced the nuclear translocational activation of NF-κB.(2) Inhibition of ER stress attenuated the nuclear translocation of AGEs-induced NF-κB by morphology.HUVECs were transfected with IRE1α siRNA and control siRNA respectively and then incubated with100mg/L AGE-BSA. NF-κB nuclear localization was observed by immunofluorescent staining. The results showed that the translocation of AGEs-induced NF-κB from the cytoplasm to the nucleus was attenuated by IRE la siRNA but not by control siRNA.It indicated that inhibition of ER stress attenuated the nuclear translocation of AGEs-induced NF-κB by morphology.4. The effect of oxidative stress on AGE-BSA-induced ER stress(1) The effect of oxidative inhibitors on expression of GRP78induced by AGEsHUVECs were incubated until the cells are60～80%confluent and then transfected with Nox4siRNA or control siRNA respectively for48h. Total proteins were collected and the expression of target molecule, Nox4, was monitored by western blotting. The results showed that Nox4protein was decreased by Nox4siRNA compared to control group but there was no difference between control siRNA and control group. It demonstrated that Nox4protein was down-regulated by Nox4siRNA.HUVECs were pretreated with Nox4siRNA or GSH respectively, and then administrated with100mg/L AGE-BSA for12h. Total proteins were collected and expression of GRP78was determined by western blotting. The results showed that AGEs-induced GRP78raising was attenuated by Nox4siRNA (P=0.014) and GSH (P=0.005) compared to AGEs group. Nevertheless, it maked no difference between control siRNA and AGEs group. It demonstrated that the increase of AGEs-induced GRP78was attenuated by oxidative inhibitors.(2) The effect of oxidative inhibitors on phosphorylation of IRE1α induced by AGEsHUVECs were pretreated with Nox4siRNA, GSH, and then administrated with100mg/L AGE-BSA for12h. Total proteins were collected and phosphorylation of IRE1α was tested by western blotting. The results showed that AGEs-induced IRE1α phosphorylation was attenuated by Nox4siRNA (P=0.041) and GSH (P=0.001) compared to AGEs group. Nevertheless, it maked no difference between control siRNA and AGEs group. It revealed that the phosphorylation of AGEs-induced IRE1α was attenuated by oxidative inhibitors.5. The effect of oxidative stress inhibition on the nuclear translocation of AGEs-induced NF-κB by morphologyHUVECs were transfected with Nox4siRNA or GSH respectively, and then stimulated with100mg/L AGE-BSA for12h. NF-κB nuclear localization was observed by immunofluorescent staining. The results showed that the translocation of AGEs-induced NF-κB from the cytoplasm to the nucleus was attenuated by Nox4siRNA or GSH but not by control siRNA. It indicated that inhibition of oxidative stress attenuated the nuclear translocation of AGEs-induced NF-κB by morphology.Conclusions:1. AGE-BSA induced ER stress in HUVECs in time-and concentration-dependent manners. 2. Inhibition of ER stress via down-regulation of IRE la expression attenuated the activation of NF-κB induced by AGEs.3. AGE-BSA induced ER stress via oxidative stress pathway.