P2Y(2)R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production.
Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y2 receptor (P2Y2R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y2R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y2R agonists ATP/UTP for 1h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y2R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y2R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y2R-mediated ROS production. Treatment with oxLDL for 24h induced P2Y2R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y2R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y2R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y2R or RAGE, suggesting that P2Y2R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y2R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-701, Korea.
So Young
SY
Eun
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-701, Korea.
Sang Won
SW
Park
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-701, Korea.
Jae Heun
JH
Lee
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-701, Korea.
Ki Churl
KC
Chang
Department of Pharmacology, School of Medicine, Institute of Health Sciences, Gyeongsang National University, Jinju 660-701, Korea. Electronic address: hyejungkim@gnu.ac.kr.
Hye Jung
HJ
Kim
D000255:Adenosine Triphosphate / Q000008:administration & dosage; D050197:Atherosclerosis / Q000378:metabolism* / Q000473:pathology / Q000628:therapy; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D008077:Lipoproteins, LDL / Q000008:administration & dosage / Q000378:metabolism; D058990:Molecular Targeted Therapy; D018384:Oxidative Stress / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products / Q000096:biosynthesis*; D058470:Receptors, Purinergic P2Y / Q000096:biosynthesis*; D014544:Uridine Triphosphate / Q000008:administration & dosage; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis
D008077:Lipoproteins, LDL; D017382:Reactive Oxygen Species; D000067759:Receptor for Advanced Glycation End Products; D058470:Receptors, Purinergic P2Y; D019010:Vascular Cell Adhesion Molecule-1; C099252:oxidized low density lipoprotein; D018799:Intercellular Adhesion Molecule-1; D000255:Adenosine Triphosphate; D014544:Uridine Triphosphate
Adhesion molecules; Free radicals; Nucleotide; OxLDL; P2Y(2)R; RAGE; ROS