Isolation and characterization of PECAM‐1‐deficient retinal endothelial cells expressing a specific isoform of PECAM‐1

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is expressed at high levels on endothelial cells. Alternative splicing of PECAM-1 generates multiple isoforms, which differ in the length of their cytoplasmic domains. We have shown that multiple isoforms of PECAM-1 are expressed in vascular beds of different tissues and endothelial cells; and their expression is regulated during vascular development and angiogenesis. However, how these isoforms differ in their adhesive and signaling properties remains largely unknown. Previous studies using PECAM-1 antibodies have demonstrated a role for PECAM-1 in endothelial cell monolayer formation, capillary morphogenesis in Matrigel, and angiogenesis. The PECAM-1 deficient (PECAM-1 -/-) mice have been generated and show a number of vascular abnormalities during angiogenesis and inflammatory challenges. The role of PECAM-1 isoforms in these processes requires further investigation. Here we describe the isolation of conditionally immortalized endothelial cells from the retinas of wild type and PECAM-1 -/- mice. We show that PECAM-1 -/- endothelial cells are less migratory and fail to undergo capillary morphogenesis in Matrigel. Expression of PECAM-1 in these cells restores these defects in an isoform specific manner. We show that expression of the PECAM-1 isoform lacking exons 14 and 15, but not the isoform that lacks exon 15, enhances the migration and capillary morphogenesis of PECAM-1 -/- retinal endothelial cells. These results demonstrate PECAM-1 functions that are essential during vascular development and angiogenesis; and are regulated by alternative splicing of its cytoplasmic domain. This work was supported by the American Heart Association Predoctoral Fellowship 0510047Z.