The Study For Effect Of Atorvastatin On Monocyte-endothelial Cell Adhesion And Its Mechanism

Objective: In this study, tumor necrosis factor-a (TNF-a) induced cultured human umbilical vein endothelial cells (HUVEC) to be lipid peroxidation.The adhesion assay had been used to observe the adhesion between HUVEC and monocytes (U937) after the intervention atorvastatin(ATV).To further research the anti-inflammatory mechanism of ATV ,we separately explore the effect of ATV on the expression level of intercellular adhesion molecule -1 (ICAM-1) on HUVEC surface and single-molecule interactional force between ICAM-1 and its ligand CD11b.Methods: HUVEC were isolated by 0.1% type I collagenase digestion for 15-20 min at 37℃, and cultured in low serum endothelial cell medium. After passage cultivation, we used the factor VIII related antibody to identify endothelial cell. Cells were used at passage 2 to 3.1 Monocyte-endothelial cell adhesion assayHUVEC were inoculated in 96-well plates, sub-experiment:①.Control group②.TNF-a group (TNF-a10ug/L)③.TNF-a+ATV group(TNF-a10ug/L+ATV10umol/l) , (incubated with TNF-a 10ug/L for 24 hours ,and 24 hours incubation with ATV ). 25% Rose Bengal staining was to detect the adhesion between HUVEC and monocyte(U937).The value A measured by microplate reader under 570nm wavelength for each hole that directly proportional to cell adhesion qu-antity.2 The effect of ICAM-1 expression on the HUVEC surface by ATV interventionThe HUVEC were seeded into 6-well plates, grouped as above, collected cells at the end of the experiment.the cell suspension transferred into inflow-pipe, and washed cells by PBS for one time. use of the remainder of the supernatant cell suspension (about 100ul), added 10ul FITC labeled ICAM-1 monoclonal antibody to each tube.washed cells by PBS for three times. Using flow cytometry to detecte expression level of ICAM-1 for each group on the cell surface(each counting 15000 cells, the results expressed by average fluorescence intensity).3 Construct plasmid of ICAM-1-GFP and transfect COS-7The total RNA was extracted from cultured HUVEC. As pEGFP-N1 to be a template,a pair of PCR primers that one end contains BamHI restriction sites, the other end contains HindIII restriction sites was Designed and Synthesised (upstream and downstream primer sequences are as follows: ICAM -UPPER: 5'CCA AGC TTC CTC GCT ATG GCT CCC AGC AG 3 '; ICAM-LOWER: 5'CTG GAT CC A AGG GAG GCG TGG CTT GTG TGT T3').The codingregion cDNA was amplified by RT-PCR and completed theDNA sequencing.The cDNA of ICAM-1 and pEGFP-N1willbe digested by double enzyme to construct a plasmid that GFP fused with the C-terminal of ICAM-1.Then transfectedthe plasmid ICAM-1-GFP into COS-7 by lipofectamine2000,and observed the cell whether issued green fluorescence bylaser confocal microscopy.4 single-molecule force spectroscopy between ICAM-1 and CD11b detected by AFMThe AFM tips were first cleaned according to standard procedures.The cleaned tips were transferred to a solution of 1.0% (v/v) MPTMS in toluene, incubated for 2 hours at room temperature to be silanized. A polyethylene glycol spacer(20-40 nm) containing an amino-reactive group at one end and a thiol-reactive group at the other end, was used to link CD11b to the silanized AFM tips in order to keep the immobilized CD11b in a flexible state.Plasmid ICAM-1-GFP was transfected by lipofectamine into COS-7 cells, grouped:①without any drug intervention (Transfected Cell);②CD11b-modified tip incubated in 10μmol /L ATV for 1 hour (Tip+ATV);③transfected cells incubated in 10μmol/L ATV for 1 hour (ICAM-1-Cell+ATV);④transfected cells incubated in anti-human ICAM-1 monoclonal antibody 200ug/ml for 1 hour (ICAM-1-Cell+Anti).To measure the adhesion force between ICAM-1 and its ligand CD11b in living cells, the CD11b-coated AFM tip was dir-ected to the ICAM-1-expressing cells with the help of the optical and fluorescence imaging of cells with the combined AFM/fluorescence microscopy system.under the scope of the loading rate 2.0±0.5×103pN/s,the CD11b-modified AFM tip repeatedly moved at the cell surface would record manyforce curves, statistics drawn affinity between tip and cell of the Gaussian probability distribution and bonding probility.Results1 In TNF-a induced group,the adhesion between HUVEC and U937 increased significantly than the control group(P<0.05). While the intervention of ATV inhibited such cell adhesion effectively(P <0.05).2 The expression of ICAM-1 on HUVEC surface increased in TNF-a group than control group(P<0.05), while the intervention of ATV would down regulate the increase of ICAM-1 expression that TNF-a induced (P <0.05)3 DNA sequencing and double restriction enzyme digestion showed successful construction of plasmid ICAM-1-GFP . The transfected COS-7 was observed that the green fluorescent protein located on the cell membrane by laser confocal microscopy(×1000).4 The results of single-molecule force measured by AFM and the bonding probability for each group:①Transfected Cell: 42.04±0.6pN; 19.15±4.2%②Tip + ATV: 40.04±0.7pN; 20.02±2.6%③ICAM-1-Cell + ATV: 39.68±1.3pN; 23.76±2.5%④ICAM-1-Cell + Anti: 27.56±0.6pN; 15.70±3.5%. There is no significant difference in the adhesion force and the binding probability in the first 3 groups(P> 0.05), while the adhesion force in the 4th Group changed to be lower compared with the previous 3 groups.(P < 0.05).Conclusion1 TNF-a induced HUVEC to be lipid peroxidation that would increase of the adhesion between U937 and HUVEC. The intervention of ATV effectively inhibited the cell adhesion.2 There was only a small amount of ICAM-1 expressing on the normal HUVEC surface.TNF-a inducing can significantly increase the ICAM-1 expression on endothelial cells,while atorvastatin effectively inhibited such increase of ICAM-1 expression.3 DNA sequencing and double restriction enzyme digestion showed successful construction of plasmid ICAM-1-GFP . The transfected COS-7 was observed that the green fluorescent protein located on the cell membrane by laser confocal microscopy.Single-molecule adhesion force between ICAM-1 and CD11b detected by AFM on the surface of living cells was at about 40pN.4 The ATV could not directly blocking ICAM-1 or CD11b to impact the molecule interaction.The ICAM-1 monoclonal antibody that non-specific block the interaction between CD11b and ICAM-1 can reduce the value of its adhesion force.