The Signal Transduction Pathways Involve In Shear Stress-induced Expression Of IL-8 MRNA In Human Endothelial Cells

BackgroundHemodynamic forces, which include the shear stress, the circumferential stress, and the normal force due to transmural pressure, play an important role in maintaining cardiovascular homeostasis and constitute a risk factor of cardiovascular diseases such as the atherosclerosis. Shear stress, the tangential component of hemodynamic forces, can modulate endothelial functions and mediate many physiological and pathological processes in part by regulation of gene encoding for many protein including vasoactive substances, adhesion molecules, growth factors, and chemotactic factors and so on. Our previous study demonstrated that IL-8 mRNA expression was increased when cultured human umbilical vein endothelial cells(HUVECs) were treated with low laminar shear stress for 1 h, and further increased when were treated for 2 h. Obviously, it should involve in complicated signal transduction pathways.The signal transduction of forces involves in mechanocoupling, biochemical coupling and so on. To evaluate the signal transduction pathway of interleukin-8 gene in endothelial cells up-regulated by shear stress, we will investigate: 1) the role of integrins, cytoskeleton and mitogen-activated protein kinase (MAPK) signal pathway in shear stress-induced expression of IL-8 mRNA in HUVECs. 2) The possible signal molecules involve in the low shear stress-induced IL-8 mRNAexpression in HUVECs.Normally, IL-8 protein is barely secreted from any induced cells, for example, endothelial cells or leukocytes and so on, but its production is rapidly induced by a very wide range of stimuli encompassing proinflammatory cytokines, Uv, stress and so on. IL-8 should serve as hormonal stimuli and affect on the proliferation, apoptosis and the function of endothelial cells. So we will investigate the effect of the rhIL-8 on the proliferation, apoptosis of HUVECs, and on release of NO, 6-keto-PGF,o, ET-1, t-PA, PAI-1 from HUVECs. MethodsHUVECs and the endothelial cells lines (ECV304 and EA.hy926) were cultured as target cells. The flow chamber was used to study the signal transduction pathways that involved in shear stress-induced expression of IL-8 mRNA in human endothelial cells in vitro. The following experiments were performed:1. The role of integrins in shear stress-induced expression of IL-8mRNA in HUVECs.The expression of integrinavp3, &2, Pi on the membrane of HUVECs was studied with Flow Cytometry(FCM), and LightCycler? system was employed to assay the expression of IL-8 mRNA of HUVECs.2. The role of cytoskeleton in shear stress-induced expression of IL-8mRNA in HUVECs.LightCycler? system was employed to assay the expression of IL-8 mRNA of HUVECs. The results were compared among the groups treated with low shear stress (4.20 dyne/cm2), cytochalasin D (F-actin cytoskeleton-disrupting agent) or cytochalasin D+ low shear stress. FITC-phalloidin was used to label F-actin .3. The possible signal molecules involved in the low shear stress-induced IL-8 mRNA expression in HUVECs.LightCycler? system was employed to assay the expression of IL-8mRNA of HUVECs .The results were compared among the groups stimulated by low shear stress (4.20 dyne/cm2) with or without all kinds of inhibitors.4. The role of MAPK signal pathway in the expression of IL-8 mRNA in HUVECs under the stimulation by low shear stress (4.20 dyne/cm2).The phosphorylation of MAPK families, including ERK1/2 (extracellular signal regulated kinase, ERKl/2)> JNK(c-Jun N-terminal kinase), P38 was detected by Western blot analysis; Immunocyto-chemistry was employed to measure the distribution and intensity of MAPK families in cultured HUVECs exposed to low shear stress; all kinds of inhibitors and dominant-negative-P38 and RNAi about JNK were used to block MAPK pathway; LightCycler? system was employed to assay the expression of IL-8 mRNA of HUVECs.5. The effect of the rhIL-8 on the proliferation and apoptosis of HUVECs, and on release of NO, 6-keto-PGFia, ET-1, t-PA,PAI-l from HUVECs.Cultured HUVECs were treated with rhIL-8 at 50 ng/ml~200 ng/ml for 12 h,24 h or 48 h. The amount of HUVECs proliferation or apoptosis was monitored by flow cytometric (FCM) .The medium was collected to test NO content by optical density. ET-1 and 6-keto-PGFia were measured by radioimmunization. A specific enzyme-linked immunosorbent assay was employed to analyses the release of t-PA and PAI-1. Results1. The role of integrins in shear stress-induced expression of IL-8 mRNA in HUVECs.(1) FN increased the expression of integrin avp3 and integrin subunit Pi,but the expression of integrin ct2 was no significant difference between with or without FN;(2) Low shear stress up-regulated the expression of IL-8 mRNA inHUVECs, and FN seemed to improve such gene expression. GRGDNP, which blocked the binding of integrins to FN, inhibited the mRNA expression of IL-8 stimulated by low shear stress. Additionally, such increase of IL-8 mRNA was attenuated by neutralizing antibodies against integrin