| 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. Intercellular adhesion molecule-2 (ICAM-2) is member of the immunoglobulin (Ig) superfamily, abundantly expressed on endothelial blood vessels, which would enable DCs to tether to and roll along vascular EC. 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 expression of ICAM-2 and DC-EC adhesion 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 mediumreplaced with fresh culture medium for 24h prior to treatment with homocysteine. The purity of EC cultures was checked by expression of factor VIE 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.3. HUVECs were exposed to Hey at different concentration (0.01 mmol/L, 0.05mmol /L, 0.1 mmol/L, 0.25 mmol/L, 0.5 mmol/L, 1.0 mmol/L) for 24h and different time (2 h, 4 h, 6 h, 12 h, 24 h, 48 h) at the concentration of 0.5 mmol/L. Cell viability was mea- sured by MTT;Enzyme - linked immuno- sorbent assay ( ELISA) was performed to measure the secretion of ICAM-2 in the supernate of cultured HUVECs;Western blot was used to detect the expression of ICAM-2 protein;RT-PCR was used to determine the expression ofICAM-2mRNA.4. HUVECs were exposed to Hey (0.5 mmol/L), folk acid (FA, lOOumol/L), folic acid + Hcy(FH), tumor necrosis factor-alpha(TNF-a, lOng/mL), TNF-a+ Hcy(TH) for 24h. Cell viability was measured by MTT;Western blot was used to detect the expres -sion of ICAM-2 protein;RT-PCR was used to determine the expression of ICAM-2 mRNA.5. Adhesion assay: After HUVECs were exposed to Hey at different concentration and FA, FH, TNF-a,TH for 24h, CMFDA labeled DC were added to HUVECs mono -layers and coincubated for lh. Besides, Anti-human ICAM-2 Antibody(25ug/mL) was added withlabeled DC to HUVECs which were pretreated with Hey at 0.5 mmol/L(HI) and coincubated for lh. Adherent cells were immediately quantified by using Leica TCS SP Spectral Confocal Microscope.Results1. After exposure of HUVECs to Hey at the concentration of 0.5 mmol/L for different time, cell viability was no change;the secretion of ICAM-2 protein was higher than those of the control group at the time of 12 h (PO.05), and was significantly higher at the time of 24 h and 48 h (PO.01);The expression of ICAM-2 protein was higher than those of the control group at the time of 6 h(PO.05), and was the highest at 24 h (PO.01);the expression of ICAM-2 mRNA was higher than the control group at 12 h (PO.05), and was significantly higher at 24 h and 48 h (PO.01) . After exposure of the HUVECs to HC Y of different concentrations for 24h, cell viability was no change, the expression and secretion of ICAM-2 protein increased in a dose-dependent pattern, and when the concentration was 0.5 mmol/L or more, the increase was more significant (PO.01);the expression of ICAM-2 mRNA was higher than the control group at 0.05 mmol/L (PO.05), and when the concentration was 0.25 mmol/L or more, the increase was more significant(PO.01) .2. After exposure of HUVECs to Hey, FA, FH, TNF-a, TH for 24h, cell viability was no change;the expression of ICAM-2 protein and mRNA of HUVECs which were pretreated with FA, FH, TNF-a showed no statistical difference compared with the control group (P>0.05);the expression of ICAM-2 protein and mRNA of HUVECs which were pretreated with Hey and TH increased significantly compared with the control group(PO.01);but the Hey group showed no statistical difference compared with the TH group at the expression of ICAM-2 protein and mRNA of HUVECs (P>0.05).3. DC-EC adhesion result: After exposure of the HUVECs to HCY of different concentrations for 24h, the adhesion DC increased at 0.1 mmol/L and 0.25 mmol/L compared with the control group, increased significantly at 0.5 mmol/L (PO.05), and increased more significantly at 1.0 mmol/L (PO.01). The adhesion DC of group FA, FH and HI showed no statistical difference compared with the control group (P>0.05);group Hey and TNF-a increased significantly (P<0.05);group TH increased more significantly (PO.01);and compared with the group Hey and TNF-a, group TH increased more significantly (PO.01).Conclusion1. Hey may enhances DC-EC adhesion by upregulating expresson and secretion of ICAM-2 in HUVECs, which indicates a novel pathophysiological mechanism for Hey to alter endothelial cell function and promote atherogenesis.2. The adhesion of DC-EC was inhibited following pretreatment with Anti-human ICAM-2 Antibody, which suggested a pivotal role for ICAM-2 in DC-EC adhesion.3. FA could decrease the expression of ICAM-2 in HUVECs which was induced by Hey, inhibit the adhesion of DC-EC, which indcated that Hey could induce ICAM-2 expression and DC-EC adhesion specially. TNF-a had no effect on the expression of ICAM-2 in HUVECs, but it could enhance the adhesion of DC-EC, which suggested that ICAM-2 was not the only adhesion molecule inducing DC-EC adhesion, there might be other adhesion molecules inducing DC-EC adhesion.
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