Abstract 333: Platelet and Endothelial Aggregation Receptor-1 Tempers Tubulogenesis via the Akt Pathway

Abstract

Platelet endothelial aggregation receptor-1 (PEAR1) is a transmembrane receptor expressed on platelets and endothelial cells (ECs). We aim to unravel its function in ECs. qPCR analyses for PEAR1 in cultured human endothelial progenitors (blood outgrowth endothelial cells (BOECs)), in ECs from human umbilical cord (HUVECs and HUAECs) and in ECs freshly isolated from multiple vascular beds showed the lowest expression in BOECs and the highest in macro- and microvascular ECs from heart and liver. HUVECs and HUAECs showed intermediate PEAR1-expression. Staining of fast growing skin hemangiomas versus normal skin ECs revealed low expression of PEAR1 in rapidly proliferating tumor ECs. Short hairpin based miRNA lentiviral PEAR1-knockdown (shPEAR1) doubled the hybrid EA.hy926 and HUVEC EC proliferation rate compared to control lentiviral-transduced ECs. This was associated with downregulation of HES1-expression (a downstream regulator of the NOTCH-pathway), with increased baseline Akt-P levels (PI3K-activity) in shPEAR1 cells and with decreased levels of the phosphatase PTEN (PI3K-antagonist). Expression of the nuclear proliferation suppressor p21CIP1 in shPEAR1 ECs was reduced by 60±2% (mean±SEM, qPCR and Western blot), whereas the band intensity for p34/CDC2 significantly rose on Western blots (increased mitosis). PEAR1-knockdown also enhanced EC migration via reduced expression of β1- and β3-integrins both in HUVECs and EAhy926 ECs. Both enhanced proliferation and migration contributed to increased tube formation in shPEAR1 HUAECs (in vitro matrigel tube formation assays). During HUAEC tube formation, PEAR1-expression rose 5-fold, associated with a 3-fold increase of PTEN. However, PTEN-levels dropped 1,5-fold during tube formation of shPEAR1 HUAECs, compatible with increased phosphorylation levels of Akt in shPEAR1 ECs. Our study provides the first evidence that PEAR1-expression is variable in ECs from multiple vascular beds and that PEAR1 plays a suppressive role on proliferation, migration and tube formation of ECs through regulation of the PI3K/Akt/PTEN-pathway. Our data suggest a regulatory role for PEAR1 in the NOTCH-pathway via HES1. The upregulation of PEAR1 during EC maturation is compatible with this suppressive role.