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Enhancement Effects of Lysophospholipids on ICAM-1, IL-8 and MCP-1 Expression on Human Endothelial Cell
No full text水解磷酸脂 Lysophosphotidic Acid (LPA)與 Sphingosine 1-Phsophate (S1P)均為存在於人體血液循環中低分子量之脂肪酸，透過辨識與連結細胞膜上Edg族受器進而調控影響細胞的生理活性，包括刺激血管內皮細胞之複製及移行。本項實驗的研究目的在探討水解磷酸脂在人類內皮細胞中對於細胞黏著因子ICAM-1與趨化因子IL-8和MCP-1表現的影響。
在human umbilical cord vein endothelial cells(HUVECs)中，透過LPA與S1P的處理均能夠誘導ICAM-1的mRNA與蛋白質的表現，並且觀察到了時間與劑量依賴的現象。透過細胞流噬儀與西方墨點的分析，LPA與S1P刺激內皮細胞表現ICAM-1蛋白質的表現於8小時後達到最大量。更進一步的，利用前處理pertussis toxin ，Gi的專一性抑制劑及PDTC，NF-Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low molecular weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors. In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. We examined the impact of lyosophosphlipids on the expression of intercellular adhesion molecule-1 (ICAM-1), and on the production of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Incubation with both LPA and S1P enhanced ICAM-1 mRNA and protein expressions in HUVECs were in a dose- and time-dependent manner. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, prior treatment of HUVECs with pertussis toxin, a specific inhibitor of Gi, or PDTC, an inhibitor of the NFκB pathway, prevented the enhanced effect of LPA- and S1P-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of rho, had no effect on LPL-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. On the other hand, LPA and S1P enhanced IL-8 and MCP-1 mRNA expression and protein secretion in a dose- and time-dependent fashion. Maximal mRNA expression appeared at 16 h postligand treatment. By chemical inhibitors prior treatment, we found both LPA and S1P enhanced IL-8 and MCP-1 expression through a Gi-, rho- and NFContents
Abstract----------------------------------------------------------------------------Ι
Introduction-----------------------------------------------------------------------1
Material and Method------------------------------------------------------------8
Reagents and antibodies----------------------------------------------------------------8
Cell culture--------------------------------------------------------------------------------9
RT-PCR for ICAM-1, IL-8 and MCP-1--------------------------------------------10
Semi-quantitative RT-PCR-----------------------------------------------------------11
FACscan----------------------------------------------------------------------------------11
Western blotting for ICAM-1--------------------------------------------------------11
Immunofluorescence staining--------------------------------------------------------12
Confocal microscopy-------------------------------------------------------------------13
Assay for U937 adhesion to treated endothelium--------------------------------13
ELISA-------------------------------------------------------------------------------------14
Chemotaxis Assays---------------------------------------------------------------------15
Statistical analysis----------------------------------------------------------------------16
Results----------------------------------------------------------------------------17
LPA and S1P increase ICAM-1 mRNA in HUVECs----------------------------17
LPA and S1P increase ICAM-1 protein expression------------------------------18
PTx and PDTC, but not exoC3, blocked LPA and S1P effects on ICAM-1 expression--------------------------------------------------------------------------------19
LPA and S1P enhanced U937 cell adherence to treated HUVECs-----------20
Both LPA and S1P increase IL-8 and MCP-1 mRNA in HUVECs-----------21
Both LPA and S1P increase IL-8 and MCP-1 protein secretion--------------22
PTx, exoC3 and PDTC, blocked LPA and S1P effects on IL-8 and MCP-1 protein secretion and mRNA expression-------------------------------------------23
LPA and S1P up-regulate chemotactic activity of conditioned media of HUVEC-----------------------------------------------------------------------------------24
Discussion------------------------------------------------------------------------26
References------------------------------------------------------------------------32
Figures----------------------------------------------------------------------------4
Sphingosine 1-phosphate regulates inflammation-related genes in human endothelial cells through S1P1 and S1P3
No full textLysophospholipids increase IL-8 and MCP-1 expressions in human umbilical cord vein endothelial cells through an IL-1-dependent mechanism
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No full textLysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kB-dependent mechanism
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