Regulation of Endothelial Cell Barrier Function by Antibody-driven Affinity Modulation of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1)

Heng Mei,Jay M Campbell,Cathy M Paddock,Panida Lertkiatmongkol,Michael W Mosesson,Ralph Albrecht,Peter J Newman

doi:10.1074/jbc.m114.557454

Abstract

PECAM-1 is a 130-kDa member of the immunoglobulin (Ig) superfamily that is expressed on the surface of platelets and leukocytes, and at the intracellular junctions of confluent endothelial cell monolayers. Previous studies have shown that PECAM-1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity. To examine the adhesive properties of full-length PECAM-1 in a native lipid environment, we purified it from platelets and assembled it into phospholipid nanodiscs. PECAM-1-containing nanodiscs retained not only their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane- proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. The finding that the adhesive properties of PECAM-1 are regulatable suggests novel approaches for controlling endothelial cell migration and barrier function in a variety of vascular permeability disorders.

Highlights

Platelet endothelial cell adhesion molecule-1 (PECAM-1) participates in adhesion and signaling in blood and vascular cells
Previous studies have shown that PECAM1/PECAM-1 homophilic interactions play a key role in leukocyte transendothelial migration, in allowing PECAM-1 to serve as a mechanosensory complex in endothelial cells, in its ability to confer cytoprotection to proapoptotic stimuli, and in maintaining endothelial cell junctional integrity
Generation and Adhesive Properties of PECAM-1-containing Phospholipid Nanodiscs—Full-length PECAM-1 purified from human platelet concentrates and the MSP1D1 form of membrane scaffold protein (Fig. 1A) were assembled at a ratio of 1:87.5:0.05625 MSP1D1:DMPC:PECAM-1 into phosphatidylcholine-containing nanodiscs as described in “Experimental Procedures,” separated from empty nanodiscs by gel filtration chromatography (Fig. 1B), and the resulting products analyzed by SDS-PAGE to confirm purity (Fig. 1C)

Summary

Background

PECAM-1 participates in adhesion and signaling in blood and vascular cells. Results: The adhesive properties of PECAM-1 in both cellular and artificial membranes can be regulated. PECAM-1-containing nanodiscs retained their ability to bind homophilically to PECAM-1-expressing cells, but exhibited regulatable adhesive interactions that could be modulated by ligands that bind membrane-proximal Ig Domain 6. This property was exploited to enhance the rate of barrier restoration in endothelial cell monolayers subjected to inflammatory challenge. Insights provided by the knowledge that the adhesive properties of PECAM-1-containing nanodiscs are regulatable suggests novel approaches for controlling both endothelial cell migration during angiogenesis and barrier function in a variety of vascular permeability disorders

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION