Role Of G31P In Smooth Muscle Migration Of A7r5 Cells Induced By Interleukin-8

Objectives:Atherosclerosis (Atherosclerosis, AS) resulting cardiovascular and cerebrovascular diseases are the biggest killers of human health, the incidence rate in China increased year by year. Recent studies have shown that AS is an inflammatory disease, a large number of inflammatory cells and inflammatory factors associated with its occurrence and development process. Inflammatory cells involved in AS were characterized by a large number of cell adhesion and infiltration in the injury site and then release large amounts of inflammatory cytokines, chemokines so as to mediate leukocytes through the endothelium, and induce smooth muscle cells phenotype changes and then migrate into the intima. After entering intima, these cells starts swallowing lipid and format foam cells, this may accelerate the development of AS. Chemokines are a class of small molecular weight secreted proteins with chemotactic effect. It can induce leukocytes to enter intima, which plays an important role in AS. Interleukin -8 (IL-8) is a secretive monocyte/macrophage peptide, which initiates neutrophil chemotactic response by binding to its receptor CXCR1/CXCR2. IL-8 can also induce vascular smooth muscle cell migration and proliferation, and can promote neovascularization in AS, but the specific mechanism is unclear. Inhibition of IL-8 binding to its receptor provides a new idea for treatment of AS. G31P is a high affinity, non-active analogues of human IL-8 screening through human IL-8 gene site-directed mutagenesis. Recent study confirmed the G31P can binding to CXCR1/CXCR2 effectively, and have a significant effect on treatment of neutrophil-related infections. whether this inhibition effect of G31P can be shown in as well as vascular smooth muscle cells has not been studied.Methods: (1) The expression of CXCR2 mRNA was detected by RT-PCR. (2)The effect of IL-8 or G31P on A7r5 cells migration were detected by Boyden chamber. (3) The effect of G31P on IL-8 induced A7r5 cells migration were detected by Wound healing and Boyden chamber. (4) Remodeling of the actin cytoskeleton detected by immunofluorescence. (5) Concentration of calcium was detected by Calcium fluorescence Probe (Fluo-3/AM) combined with microscopic imaging system. Results: (1) The expressions of CXCR2 mRNA in A7r5 cells were revealed by RT-PCR. (2) A increase in migration cell number was observed by Boyden chamber assay with dose increase of IL-8, the groups treated with IL-8 20ng/ml or 50ng/ml shows a significant increase respectively and non-significant difference between them. Whereas the chemotactic effect on A7r5 cells migration of G31P was not shown in this study. (3) Significantly chemotactic effect of IL-8 on A7r5 cells (p<0.01 vs. control and G31P group) observed form wound healing and Boyden chamber, and this effect were inhibited by different concentrations of G31P(p<0.01). (4) The filopodia of IL-8 induced cells increased in a dose-dependent way. Pretreated with G31P can reduce filopodia formation. (5) The increase of Fluorescence intensity in A7r5 cells with IL-8(0-100ng/ml) indicated that the concentrations of calcium were significantly increased (p<0.01). Pretreated with G31P can significantly reduce the increase (p<0.01).Conclusions: (1) IL-8 can induce smooth muscle cell migration, filopodia formation and calcium concentration increase in A7r5 cells in a dose-dependent way. (2) G31P can significantly inhibit the chemotactic effect of IL-8 on A7r5 cells. (3) The effect of IL-8 on A7r5 cells probably was regulated by membrane calcium channel.