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 (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-1 (MCP-1) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-1, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S1P enhanced IL-8 and MCP-1 mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at 16 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-1 expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-1 protein secretions. Pre-incubation with AF12198, an IL-1 receptor antagonist or IL-1 functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-1 mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-1-dependent chemoattraction of monocytes toward the endothelium through an IL-1-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that 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. The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily. This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs). This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment. Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting. Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent. Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G(i), ammonium pyrrolidinedithiocarbamate and BAY 11-7082, inhibitors of the nuclear factor (NF)-kappaB pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression. However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression. In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line. The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1. These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G(i)-, NF-kappaB-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are both low-molecular-weight lysophospholipids, which promote cell proliferation, migration, and invasion via interaction with a family of specific G protein-coupled receptors. Matrix metalloproteinases (MMPs) are zinc-dependent proteolytic enzymes, which are involved in degradation of the extracellular matrix and play critical roles in endothelial cell migration and matrix remodeling during angiogenesis. Among these MMPs, MMP-2 is known to trigger cell migration. In our present study, we examined the effects of LPA and S1P on MMP-2 expression in human endothelial cells. We showed that LPA and S1P enhanced MMP-2 expression in mRNA, protein levels, and also enzymatic activity of cells of the EAhy926 human endothelial cell line. The enhancement effects occurred in concentration- and time-dependent manners. Results from real-time PCR, Western blots, and substrate gels indicated that these enhancement effects were mediated through MAPK kinase/ERK-, nuclear factor-kappaB-, and calcium influx-dependent pathways. Furthermore, we show that endothelial cell invasion of the gel was enhanced by lysophospholipids, and the induction could be prevented by an MMP inhibitor, GM6001. These observations suggest that LPA and S1P may play important roles in endothelial cell invasion by regulating the expression of MMP-2.

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