VNR for 24 h. Following stimulation, HUVECs were harvested and incubated with fluorescence conjugated anti ICAM 1, anti VCAM 1, and anti E selectin for 45 min at room temperature. After the HUVECs had been washed three times, their immunofluorescence intensity was analyzed by flow cytometry using a Becton Dickinson FACScan flow cytometer. Adhesion Assay HUVECs at 1 9 105 cells/ml were cultured Cediranib AZD2171 in 96 well plates. HUVECs were incubated with VNR for 24 h. The medium was then removed, and 0.1 ml/well of THP 1 cells were added to fresh RPMI. The cells were allowed to adhere at 37 C for 1 h in a 5% CO2 incubator. Plates were washed three times with M199 to remove the non adherent cells. The number of adherent cells was estimated by microscopic examination, and the cells were then lysed with 0.
1 ml 0.25% Triton X 100. Fluorescence intensity was measured with a fluorescence microplate reader calibrated for excitation at 485 nm and for emission at 538 nm. Assay for IL 8 Secretion HUVECs were seeded in 24 well plates at 0.5 9 105 cells. After 2 days, HUVECs were incubated with VNR for 24 h. At the end of the VNR incubation period, cell supernatants were removed and assayed for IL 8 concentration using an ELISA kit obtained from R&D Systems. Data are expressed as ng/ml for duplicate samples. Statistical Analyses Results are expressed as mean SEM. Differences between groups were analyzed using one way ANOVA followed by Bonferroni,s post hoc test. A P value of 0.05 was considered statistically significant.
Results VNR Induced Dephosphorylation of AMPK a, Phosphorylation of PKC ab, and PKC a Activity AMPK can reverse and alter many cellular pathways to protect against oxidative injury. We assumed that VNR induced endothelial cell dysfunction was caused by repression of AMPK phosphorylation. To verify our hypothesis, the protein expression level of phosphorylated AMPK was determined using a western blotting assay. As shown in Fig. 1a and b, treatment of HUVECs with VNR for 1 h led to an attenuation of phosphorylated AMPK a in a dose dependent manner. Furthermore, previous studies have shown that PKC isoforms play a key role in the regulation of NADPH subunit expression and, in particular, the translocation of p47phox from the cytosol to the membrane, and AMPK a can inhibit ROS production through suppression of protein kinase C, which in turn prevents the activation of NADPH oxidase.
We, therefore, focused our attention on determining whether VNR facilities PKC phosphorylation and activation in human endothelial cells. As shown in Fig. 1c and e, VNR markedly increased phosphorylation of PKC ab and PKC a activity after a 1 h exposure. Pre treatment of AICAR, one agonist of AMPK, significantly mitigated VNR promoted phosphorylation of PKC ab and PKC a activity, indicating that PKC is implicated in VNR induced endothelial cell injury and mainly go through AMPK. VNR Induced Membrane Assembly of NADPH A previous study revealed that VNR induces ROS formation in endothelial cells, thereby facilitating endothelial cell apoptosis. We proposed that VNR facilitates ROS production mainly by promoting PKC phosphorylation and activating NADPH oxidase. DPI, an inhibitor of NADPH oxidase, was used to prove our hypothesis. In endothelial cells, the NOX family of NADPH o