Local Dynamic Recruitment of Endothelial PECAM-1 to Transmigrating Monocytes

Abstract

One of the critical events in early atherosclerosis is the recruitment of blood monocytes to proatherogenic vascular regions and subsequent transendothelial migration. This process involves a multi-step interaction between monocytes and endothelial cells (EC) including rolling, firm adhesion, locomotion, and transmigration. While the molecular mechanisms underlying rolling and firm adhesion have been well documented, the final step, transmigration, is still poorly understood. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is considered to be one of the key molecules in the transendothelial migration of monocytes. To investigate the role of PECAM-1 in monocyte diapedesis, we expressed a green fluorescent protein (GFP)-labeled construct in interleukin-1beta-stimulated human umbilical vein endothelial cells. Labeling PECAM-1 with GFP at its intracellular C-terminus could keep the molecular interactions with monocyte PECAM-1 intact. Three-dimensional molecular imaging in live cells demonstrated that endothelial PECAM-1 dynamically redistributed during paracellular transmigration, to surround monocytes transmigrating through both bicellular and multi-cellular junctions. Within 20 minutes of transmigration, PECAM-1-GFP was recruited at transmigration sites to induce a significant increase, with a concomitant reduction in the neighboring cytoplasm. These dynamics were not observed during transcellular diapedesis. At bicellular junctions, PECAM-1 recruitment did not occur in the region without transmigration (but with adhesion on endothelium), although it did at multicellular junctions, nor did it appear to occur at either junction type when monocyte adhesion was blocked. These data show that monocyte adhesion can trigger PECAM-1 recruitment from sub-junctional cytoplasm preferentially to multicellular endothelial junctions. Subsequent transmigration induces PECAM-1 redistribution and further recruitment at multicellular and bicellular, but not at transcellular sites, which may support chronic paracellular transmigration.