Abstract
Molecular mechanism on leukocyte transendothelial migration (TEM) in a variety of pathogenesis such as atherosclerosis is mostly unclear. We have previously reported that oxidized LDL, one of the essential players in atherogenesis, induced the expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on endothelial junctions, and specifically promoted monocyte TEM. In the present study, to shed light on the specific role of the transmembrane protein PECAM-1 in monocyte TEM, we constructed the recombinant vector expressing red fluorescent protein DsRed fused to the C-terminus (intracellular side) of PECAM-1. This mode of fusion could keep the extracellular domain of the molecule intact, allowing interaction with other cells during TEM. The vector, which was constructed with general procedure (primer design, RNA extraction, reverse transcription, PCR amplification, vector ligation, clone selection, plasmid purification, and DNA sequence) was transfected into human umbilical vein endothelial cells (HUVECs) using nucleofection technique. The expressed fusion protein was confirmed to migrate as a single molecular mass with the expected size of 160 kDa (western blotting), and localize at cell-cell contact, which is the same distribution with endogenous PECAM-1 (Immunofluorescence). We are now studying the live-cell dual imaging of endothelial (DsRed) and monocyte (GFP) PECAM-1 during TEM with two-photon excitation microscopy. [J Physiol Sci. 2007;57 Suppl:S206]
