Effects Of Homocysteine On Dendritic Cell Adhesion And Transmigration By Vascular Oxidative Stress

ObjectiveAtherosclerosis(AS) begins as an inflammatory immunological disease. The dendritic cells(DC)-endothelial cells(EC) interactions play a central role in early immune response of AS. Although homocysteine (Hcy) is a recognized independent cardiovascular risk factor, the mechanisms by which it alters the physiology of the vascular wall remain largely unknown. Therefore, we investigate the effect of Hcy on reactive oxygen species (ROS) generation of EC and DC adhesion or transmigration with cultured Human Umbilical Vein Endothelial Cells (HUVECs), to explore the potential mechanism of Hcy on atherogenesis.Methods1. HUVECs Culture: HUVECs were isolated by collagenase type I, digestion of human umbilical veins by means of standard techniques, and cultured in M199 medium with 15% fetal calf serum(FCS) and epidermal growth factor (EGF, 10 ng/ml). The medium was refreshed every 2-3 days. Cells were grown to near confluence and the medium replaced with fresh culture medium for 24h prior to treatment with homocysteine. The purity of EC cultures was checked by expression of factor Ⅷ and found to be greater than 99% positive.2. DC Culture: White blood cell suspension which was contributed by normal donors was contained from blood center of Zhejiang Province. Peripheral blood monouclear cells(PBMCs) were harvested from interface with lymphocyte separating medium. CD14~+ cells were separated by means of immunomagnetic technique (MACS) with >98% purity. Then those cells were plated in RPMI 1640 containing 15% fetal bovine serum supplemented with granulocyte-macrophage colony- stimulating factor (rhGM -CSF, 20ng/ml) and interleukin-4 (rhIL-4, 2ng/ml). Cultures were fed every other day by half. On day 5, buoyant cells were harvested as immature DC for adhesion assay or transmigration assay.3. HUVECs were exposed to Hcy at different concentration (0.01 mmol/L, 0.05mmol /L, 0.1 mmol/L, 0.5 mmol/L, 1.0 mmol/L) for 24h. In some experiments, HUVECs were pre-incubated with SOD (200U/mL) for 1h and then co-incubated with Hcy (0.5 mmol/L) for another 24h. One group of ECs were incubated only with fresh culture medium as control.4.Immunofluorescence test: After supernatant fluids were removed and the EC monolayers were washed with Hanks balanced salt solution (HBSS), pretreated ECs were incubated with 10 μmol/L HE at 37°C for 45min and then washed twice with HBSS. The fluorescence intensities were immediately detected in several regions of interest using a inverted biological microscope controlled by MetaMorph software. ROS generation of pretreated ECs was analyzed in 6 independent high-power fields by Meta Imaging Series of MetaMorph software for each experiment. Values obtained are expressed as arbitrary fluorescent units relative to control conditions5. Adhesion assay: After HUVECs were exposed to different concentration of Hcy with or without SOD, CMFDA labeled DC were added to HUVECs mono-layers and coincubated for 1h. Adherent cells were immediately quantified by using Leica TCS SP Spectral Confocal Microscope. The average number of DCs adhering to ECs was calculated and expressed as the DC/EC binding ratio.6. Transmigration assay: Fibronectin-coated transwell inserts were used to culture endothelial monolayers in 12-well plates. Prelabeled DCs were added to the upper chamber after the EC monolayers were treated and rinsed with serum-free HBSS, and 200 μL serum-free medium, supplemented with 100 ng/mL monocyte chemotactic protein-1 (MCP-1), was added to the lower chamber. After 8 hours' co-incubation at 37°C, transmigration was stopped and transmigrated cells in the lower chamber were immediately counted with the use of Synergy HT Multi-Detection Microplate Reader. Relative fluorescence intensity has been shown to increase linearly with the cell number. The migration index was calculated on the basis of the ratio between the absolute numbers of migrated cells to DC/EC binding ratio.Results1. Effect of Hcy on endothelial ROS generation: Incubation of HUVECs with increasing concentrations of Hcy resulted in a dose-dependent and significant increase in intracellular fluorescence intensity which meant an increased generation of ROS. From 0.05 to 1.0 mmol/L, Hcy enhanced endothelial ROS generation compared with the control group of untreated ECs (P<0.05 at 0.05 mmol/L or P<0.01 at higher concentrations) While pre-incubated with SOD before Hcy significantly reversed its effect on endothelial ROS generation (P<0.01).2. Effect of Hcy on adhesion of DC and EC: The adhesion of DC on ECs activated by Hcy was markedly increased from the concentration of 0.1 mmol/L compared with nonactivated ECs (P<0.05), and more significantly at the higher concentration of Hcy (P<0.01). While pretreated with SOD before Hcy 0.5 mmol/L markedly ameliorated the effect of Hcy on DC adhesion (P<0.01).3. Effect of Hcy on transmigration of DC through EC: Increasing concentrations of Hcy enhanced DC migration from 0.1 mmol/L (P<0.05) to 0.5 or 1.0 mmol/L (P<0.01)compared with untreated ECs, while SOD significantly inhibited DC transmigrating through endothelium (P<0.01).Conclusion1. Hcy induces vascular oxidative stress which leads to endothelial dysfunction, promoteing DC adhesion and transmigration, which may indicate a novel pathophysiological mechanism for Hcy to promote atherogenesis.2. SOD, one of important cellular antioxidant enzymes which degrade ROS may ameliorat Hcy induced vascular oxidative stress and DC adhesion and transmigration.