An Experimental Model for Studying Molecular Behavior of Platelet-Endothelial Cell Adhesion Molecule-1 during Mechanical Interactions between Monocytes and Vascular Endothelial Cells

Monocyte accumulation in the arterial intima is a hallmark in early atherosclerosis. Monocyte extravasation involves sequential processes including rolling, adhesion, and transmigration across vascular endothelial cells (ECs), where abundant mechanical interactions between these two cells exist. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a junctional protein expressed on both cells, and participates in paracellular transmigration via homophilic binding between these cells, while PECAM-1 binds homophilically between neighboring ECs in the absence of monocytes. During monocyte transmigration, PECAM-1-bearing membrane can be recruited to the transmigration spot from intracellular pools. The mechanism by which PECAM-1 binding between ECs in control state is switched to that between monocytes and ECs during transmigration, and its relationship with recruited PECAM-1 remain unclear. In this study, we built an experimental model in vitro for studying molecular behavior of PECAM-1 during monocyte transmigration. A plasmid vector containing PECAM-1 tagged with green (GFP) or red (DsRed) fluorescent protein was constructed, and separately transfected into ECs. The mixture of both transfectants in culture achieved a monolayer that contains an intercellular PECAM-1 boundary between PECAM-1-GFP- and PECAM-1-DsRed-expressing cells which is visualized as yellow in merged image. Using this model together with confocal laser scanning microscope-based system, molecular behavior of PECAM-1 on neighboring ECs during monocyte transmigration was observed in live cells. This model can be essential to directly visualize binding/dissociation state of PECAM-1 between neighboring ECs during monocyte trans-endothelial migration in relation to mechanical monocyte-EC interactions.