Abstract
Abstract Placenta Endothelial Protein 1 (PEP1) is a novel endothelial-specific protein which was identified by Serial Analysis of Gene Expression (SAGE) database mining in our previous study. It was predicted to be a matrix-associated protein containing eleven Thrombospondin typeⅠ repeats (TSR), one CD36-binding domain and one RGD motif. These features implied the role of PEP1 in cell-to-cell and cell-to-ECM interactions. To further explore the physiological function of PEP1, we conducted in vitro and in vivo experiments. In our other in vivo studies, we chose Tg(fli1-EGFP)y1 transgenic zebrafish as the animal model system to monitor vasculature formation during embryonic development. We observed aberrant intersegmental vessels, atrophic aortic arches, detrimental blood circulation, hindbrain ventricular edema and pericardial edema in the PEP1-knockdown morphants by injecting respective morpholino antisense oligos into zebrafish embryos. These results indicated the importance of PEP1 during angiogenic development. Therefore, in order to investigate the effect of PEP1 on endothelial cells, we took advantage of human umbilical vein endothelial cells (HUVEC) and perform further assays on cellular level. Our in vitro experimental results showed that the truncated fragment of PEP1 c-terminal end (PEP1-CTE) inhibited HUVEC migration with no effect on cell proliferation. Furthermore, immunocytochemistry staining (ICC) exhibited a co-localization pattern of PEP1 and paxillin (PXN) in HUVECs, suggesting that the cellular function of PEP1 is associated with PXN-mediated signal transduction pathways. According to a published genome-wide yeast two-hybrid screening of novel protein interactions, we came across the clues that PEP1 may interact with a recently identified cytosolic protein, development and differentiation enhancing factor 2 (DDEF2), which was known to bind paxillin. To confirm this finding, we demonstrated the interaction of PEP1 and DDEF2 by ELISA. Taken together, our results suggested that PEP1 is a novel component protein in the DDEF2/Paxillin signal transduction pathway, which highlights the important role of PEP1 in vascular development and angiogenesis process.