Enhancement Of Endothelial Cell Barrier Function By Antibody-Driven Affinity Modulation Of PECAM-1

Endothelial integrity is a critical parameter of hemostasis, and loss of barrier function contributes to the etiology and pathogenesis of both autoimmune and infectious disease. PECAM-1 is a cellular adhesion and signaling receptor comprised of six extracellular immunoglobulin (Ig) – like homology domains, a short transmembrane domain, and a 118 amino acid cytoplasmic domain that becomes serine and tyrosine phosphorylated upon activation. PECAM-1 is a major constituent of the endothelial cell intercellular junction, where up to 2 x 10⁶ molecules concentrate upon cell-cell contact. Previous studies have shown that PECAM-1–PECAM-1 homophilic interactions mediated by N-terminal Ig domain 1 (IgD1) are required for border localization, and contribute importantly to steady-state endothelial cell barrier stability as well as the ability of the vascular endothelium to recover, both in vitro and in vivo, following inflammatory or hemostatic challenge. To examine whether the homophilic adhesive properties of PECAM-1 might be subject to regulation, we prepared phospholipid bilayer nanodiscs containing purified, full-length PECAM-1 and examined their ability to bind intact PECAM-1-expressing cells. Preliminary transmission electron microscopy studies indicated that PECAM-1-containing nanodiscs harbored a single, high-density refraction shadow, suggesting the presence of a single inserted PECAM-1 protein per nanodisc. Binding of these nanodiscs to either human umbilical vein endothelial cells or to PECAM-1-transfected HEK 293 cells required IgD1, as Fab fragments of the IgD1-specific monoclonal antibody (mAb), PECAM-1.3, completely blocked their binding. Interestingly, Fab fragments of mAbs PECAM-1.2 or 4G6, which bind closely-spaced epitopes within membrane-proximal IgD6, were found to enhance PECAM-1 nanodisc binding by 200-300%, suggesting that they induced a conformational change leading to a high-affinity form of PECAM-1. In support of this notion, rotary-shadowed electron micrographs of the purified PECAM-1 ectodomain revealed an ensemble of straight and bent molecular isoforms, reminiscent of conformational isomorphs that exist among members of the integrin family of adhesion receptors. Finally, to determine whether regulatable affinity modulation of this Ig superfamily member might be exploited to enhance or reinforce endothelial cell-cell borders, endothelial cell monolayer resistance was quantitatively measured using Electric Cell-substrate Impedance Sensing (ECIS), a real-time reporter of barrier function. As predicted, IgD1-specific mAb PECAM-1.3 delayed barrier recovery following thrombin-induced disruption of the endothelial cell monolayer, while IgD6-specific mAb PECAM-1.2 markedly enhanced the rate of barrier restoration. Both antibodies were without effect on endothelial cell monolayers in which PECAM-1 had been knocked down using a lentivirally-introduced PECAM-1 siRNA. Taken together, these data demonstrate that the homophilic binding affinity of PECAM-1 can be enhanced by IgD6-specific antibodies, and suggest a novel therapeutic modality for treating vascular endothelial injury-related disorders.