Research On The Molecular Mechanism Of RhoA Mediated Endothelial Cell Migration Induced By IL-8

BackgroundCell migration is an essential process for normal development andhomeostasis that can also contribute to important pathologies. Clinicalresearches demonstrate that both wound healing produced by vesselsurgery and reendothelization of the endothelial injury area are achievedprimarily by the migration of endothelial cells from the adjacent areaswith an intact endothelium. The migration of endothelial cell also playsan important role in vessel remodeling process. For example, it is a majorfactor contributing to the restenosis that following angiogensis in tissueregeneration, vessel bypass surgery, balloon angioplasty and stentplacement is the injury of endothelium, which normally forms anon-thrombogenic lining for the arterial wall. A rapid reendothelization ofthe arterial wall is critical for the prevention of the thrombosis andmaintenance of lumen patency. Even in some chronic diseases such ascancer, atherosclerosis, and chronic inflammation fibrosis always comewith specific cell migration. In the invasion of malignant tumor, with theendothelial cell migration, new vessels are shaped in the tumor entity to supply the sufficient nutrition for the proliferation of tumor cell.Angiogensis is a process that endothelial cells migrated and proliferatedfrom built-in vessel to build new vessels through gemmation ornon-gemmation in physiological and pathologic conditions. The processof cell migration is divided into three steps: cell proliferation, migrationand lumina formation. Among these steps, cell migration is one of themost important stages.In physiological and pathologic processes, many inductive effectorscan promote the migration of vascular endothelial cells. As ainflammatory factor, IL-8 is a chemotaxis factor that belongs to CXCfamily, which is purified from human monocytes culture supernatantstimulated by Lipopolysaccharie (LPS) or Phytohaemagglutinin (PHA).It was discovered by Yoshimura in 1987, and secreted by monocytes andendothelial cells. IL-8 is a key signal molecule to induce chemotaxis ofmany inflammatory cells, participating the pathologic process of manynon-special inflammatory. However, many studies manifested that IL-8can also activate endothelial cells. It is found that there is high expressionof IL-8 in the local of atherosclerosis or at the lesion of endothelial cells.IL-8 is believed to participate the whole course of atherosclerosisformation which is initiated by the migration of endothelial cell.Recently, more attentions have been paid to investigate signaltransduction of cell migration. It is found that cell migration beginswhen a cell responds to an external signal by polarizing and extending aprotrusion in the direction of movement. The formation of adhesioncomplexes functions to stabilize the protrusion by attaching it to thesubstratum on which the cell is migrating. These adhesions, which serveas traction points for migration, initiate signals that regulate adhesiondynamics and protrusion formation. Contraction then moves the cell bodyforward and release of the attachments at the rear. As the cell retracts, the cycle is completed. It involves in cells framework and cells adherentrelated assemble and space position change in cell migration. Small Gprotein Rho family is one of the main regulation factors in reassemble ofactin cytoskeleton which plays an important function in coordinating cellsmigration. It is found that matrix instantly provided information to Rhoprotein of migrating cells in the research of integrin signal path, thusinfluence cell migration. Other researches manifest that the ablation ofcoronary artery atherosclerotic plaque can be induced by inhibiting theactivity of RhoA kinase. It is suggest that RhoA protein may be one of thecritical molecules in IL-8 induced endothelial cell migration.The purpose of this study is to investigate the mechanism of themigration of endothelial cell induced by chemokine IL-8. On the basis ofpreliminary experiments, we observed the effect of RhoA on the processof cell migration. In addition, this study is aimed to reveal thecellular-molecular mechanism in relation to the neoangiogenesis ofmalignant tumor and generate the new solutions of cancer diagnosis andprevention of malignant tumor.MethodsWe selected the EA. hy 926 cell line as seed cells:1. The transwell chamber motility assay was applied to observe themigration of endothelial cells induced by different concentrationof IL-8 (0, 50, 100, 200 ng/ml).2. Endothelial cells were infected, respectively, with theconstitutively active forms of RhoA (RhoA63L), the dominantnegative forms of RhoA (RhoA188A), and the wild type RhoA(RhoA wt) by Lipofectamaine 2000 reagant. The positive cloneswere obtained by G418 selection.3. The mRNA and protein expression of RhoA and active RhoA in infected cells were measured by RT-PCR, Pull-down and Westernblot, respectively.4. 5×10~4 endothelial cells were added to the inserts of transwellfilters. After the addition of IL-8 (100 ng/ml) to the bottom wells, the cells were incubated for 4h at 37℃in a tissue cultureincubator. When time is over, the transmigrated cells werecounted on a microscope. The results were compared amonggroups RhoA63L, RhoA188A, RhoA wt, and control group.Results1. The results demonstrated that the migration of cells was increasedsignificantly under different IL-8 concentrations, while the besteffect occurred when IL-8 concentration was 100 ng/ml.2. The constitively infected cell line is obtained. These cell linescontained the plamid RhoA (RhoA63L), the dominant negativeforms of RhoA (RhoA188A), and the wild type RhoA(RhoA wt)respectively.3. RT-PCR certified the expression of RhoA mRNA in transfectedcells, Western blot confirmed the RhoA protein in all threetransfected groups had an increase in different degrees, Pull-down assured the actived RhoA protein.4. Under the stimulation of IL-8, the ability of RhoA63L group cellmigration is high than other groups, but the ability of RhoA188Agroup cell migration is inhibited obviously.Conclusion1. As a new cell chemotactic factor, IL-8 can not only induce themigration of inflammation cells, but also induce vascularendothelial cell migration efficiently. It can elevate the mobility of endothelial cell significantly at the concentration of 100 ng/ml.2. As a member of small G protein familiy, RhoA functions adecisive effect in the process of cell migration. When theexpression of active RhoA protein is up-regulated, the migrationof endothelial cells elevated.