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

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.