Altered Expression Pattern of Alternatively Spliced PECAM–1 Isoforms in Retinal Vasculature and Endothelial Cells of Thrombospondin–1 –/– Mice

Abstract

Abstract: : Purpose: We have previously shown that thrombospondin–1 (TSP1) and PECAM–1 are components of a regulatory switch whose reciprocal regulation in the endothelial cells (EC) promotes an angiogenic or a differentiated, quiescent phenotype. The physiological role TSP1 plays in modulation of PECAM–1 expression and function during vascular development and angiogenesis remains largely unknown. Methods: Using RT–PCR and RNA prepared from retinas of wild type and TSP1 –/– mice at different postnatal days of development in room air or during oxygen–induced ischemic retinopathy, we determined the expression pattern of alternatively spliced PECAM–1 isoforms. The expression pattern of alternatively spliced PECAM–1 isoforms in retinal EC prepared from wild type and TSP1 –/– mice was also investigated. Using Western blot analysis of total cell lysates and PECAM–1 immunoprecipitation, we identified proteins that interact with PECAM–1 in retinal EC and studied the down–stream consequences of these interactions on EC migration in scratch wound assays. Results: Here we demonstrate that PECAM–1 undergoes alternative splicing in its cytoplasmic domain, generating eight isoforms in the retinal vasculature of wild type and TSP1–/– mice. All PECAM–1 isoforms examined contained exon 13. The frequency of PECAM–1 isoform(s) containing exon 14 was significantly higher during early stages of retinal vascularization and decreased during later stages of retinal vascularization in wild type mice. In contrast, the frequency of exon 14 containing PECAM–1 isoform(s) did not significantly change during retinal vascularization in TSP1 –/– mice. They consistently expressed a higher number of isoforms with exon 14 during later stages of retinal vascularization. The higher level of PECAM–1 isoforms with exon 14 was also observed in cultured TSP1 –/– retinal EC compared to wild type retinal EC. This was consistent with increased amounts of Src and SHP2 associated with PECAM–1, and enhanced migration and proliferation in TSP1 –/– retinal EC. Conclusions: These data suggest PECAM–1 signaling in the endothelium is modulated by its alternative splicing during retinal vascular development and angiogenesis and is impacted by TSP1 expression.

Keywords: retinal neovascularization • retinopathy of prematurity • cell-cell communication