Mechanism Of Effects Of ICAM-1and CX3CL1 On The Cytoskeleton In Endothelial Cells

Objective:To investigate the effects of ICAM-land CX3CL1 seperately on cytoskeleton in HUVECs, to evaluate the underlying mechanism.Method:(1) Observe the effects of ICAM-land CX3CL1 seperately on the cytoskeleton in HUVECs:HUVECs were starved for 12 hours; ① HUVECs were stimulated with 1 nMICAM-1 for 0、30、60、120、180 minutes respectively after pretreatment with 10μM Fg for 30 minutes;② HUVECs were stimulated with 10 nM CX3CL1 for 0、30、60、120、180 minutes respectively; Cells were fixed with 4% paraformaldehyde and stained with Alexafluor(?) 488 phalloidin for characterizing the frame of cytoskeletal arrangement. Fluorescence microscope was used to monitor the changes of F-actin.(2) Investigate the effects of ICAM-1 and CX3CL1 on mitogen-activated protein kinase signaling pathway in HUVECs:HUVECs were starved for 12 hours; ① HUVECs were stimulated with 1 nM ICAM-lforo、5、15、30、60 minutes respectively after pretreatment with 10μM Fg for 30 minutes; ② HUVECs were stimulated with 10 nM CX3CL1 for o、1、5、15、30 minutes respectively; ③HUVECs were stimulated with lnM ICAM-1for 5 minutes and 15 minutes or 15 minutes and 30 minutes respectively after pretreatment with 10 μM Fg and 5μg/mL anti-ICAM-1 antibody for 30 minutes; ④ HUVECs were stimulated with 10 nM CX3CL1 for 1 minutes and 5 minutes respectively after pretreatment with 5μg/mL anti-CX3CRlantibody for 30 minutes. Westrn blot was used to measure the phosphorylated p38、ERK1/2 and JNK1/2 and the total p38、 ERK1/2 and JNK1/2.(3) Investigate the mechanisms of ICAM-1 and CX3CL1 on the cytoskeleton changes in HUVECs:HUVECs were starved for 12 hours; ① HUVECs were pretreated with 30μM SB203580, PD98059 and SP600125 respectively for 30 minutes, which were used as standard inhibitors for p38. ERK1/2 and JNK1/2, HUVECs were dealt with lnM ICAM-lfor 180 minutes after pretreatment with 10μM Fg for 30minutes; ② HUVECs were pretreated with HUVECs were dealt with 10 nM CX3CL1 for 120 minutes after pretreatment with 30μM SB203580, PD98059 and SP600125 respectively for 60 minutes. F-actin disorganization was detected by fluorescence microscope after the cells were fixed and stained.Results:(1) The blank control cells showed that smooth and continuous staining for F-actin along with the intercellular borders of adjacent cells to form dense peripheral band. HUVECs stimulated with ICAM-1 for 30 minutes had little change to be observed; after 60 minutes stimulation with CX3CL1, F-actin began to have became discontinuous and rough on the edge of the cell, and stress fiber began to appear in the cytoplasm; after 120 minutes thin F-actin staining with organization of thin actin cables that can be seen stretching the length of the cell; after 180 minutes, F-actin staining continued to increase in intensity. HUVECs stimulated with CX3CL1 for 30 minutes, F-actin began to have became discontinuous and rough on the edge of the cell, and stress fiber began to appear in the cytoplasm; after 60 minutes thin F-actin staining with organization of thin actin cables that can be seen stretching the length of the cell; after 120 minutes F-actin staining continued to increase in intensity, after 180 minutes, stress fiber was decreased and shorten.(2) Prominent enhancements of phosphorylated ERK1/2 and JNK1/2 were detected starting as early as 5 minutes and peaking after 15 minutes of stimulation with lnM ICAM-1, but a prominent enhancement of p38 was detected starting as early as 5 minutes and peaking after 30 minutes of stimulation with lnM ICAM-1. Prominent enhancements of phosphorylated p38s ERK1/2 and JNK1/2 were detected starting asearly as 1 minutes and peaking after 5 minutes of stimulation with 10 nM CX3CL1. The up-regulation of phosphorylated p38、ERK1/2 and JNK1/2 were inhibited by 5μg/mL anti-ICAM-1 antibody and 5μg/mL anti-CX3CR1 antibody respectively.(3) The morphological alteration of cytoskeleton were abolished by 30μM SB203580 in HUVECs stimulated with ICAM-1 for 180 minutes, and abrogated by 30μM SB203580 and PD98059 respectively in HUVECs stimulated CX3CL1 for 120 min.Conclusion:(1) ICAM-1 may induce a time-dependent cytoskeleton disorganization by a p38 dependent mechanism in HUVECs.(2) CX3CL1 may induce a time-dependent cytoskeleton disorganization by a p38 and ERK1/2 respectively dependent mechanism in HUVECs.