摘要
藉由serial analysis of gene expression (SAGE)的方法，我們發現了一個在內皮細胞具有高度表現量的新穎蛋白質－胎盤內皮蛋白質 1(PEP1)。而經由PEP1已被預測出具有11個凝血酶敏感蛋白-1 Thrombospondin type-1 (TSP-1)重複，一個CD36結合區以及一個RGD序列的結果顯示：其生理功能必然與細胞移動以及細胞-胞外基質交互作用有相關性。為了進一步了解PEP1之生理功能，我們同時利用活體內及體外研究的方法來驗證我們的假設。在之前的研究中，我們以Tg(fli1-EGFP)y1品系基因轉殖斑馬魚做為實驗動物來研究胚胎發育過程中的血管內皮細胞。經由注射反義寡核苷酸(morpholino)進入斑馬魚胚胎來降低PEP1蛋白質表現後，我們發現發育過程中出現了不規則的體節血管(ISV)發育、萎縮的動脈弧、血流循環不正常以及後腦及心臟周邊水腫的現象。這些結果顯示了PEP1在胚胎發育中的血管新生過程扮演了重要的角色。為了進一步研究PEP1對於血管內皮細胞的影響能力，我們利用人類靜脈內皮細胞(HUVEC)來進行細胞層次的實驗。從實驗結果得知，我們發現PEP1蛋白質C端尾端胜肽片段(CTE)具有抑制HUVEC移動的能力，但不會影響其生長繁殖能力。同時，免疫螢光細胞染色結果顯示，PEP1在細胞中的表現區域與paxillin相似，暗示了PEP1可能參與paxillin相關的訊號傳遞過程。同時，根據文獻，我們猜測PEP1可能會與一個表現於細胞質內的蛋白質DDEF2有交互作用，此蛋白目前已知會與paxillin結合。利用酵素免疫分析法(ELISA)，我們驗證PEP1及DDEF2的交互作用以及確認兩蛋白間的結合區域。綜合以上實驗結果，我們的研究支持了我們的假說，也就是PEP1與DDEF2有交互作用，進而可能參與了paxillin相關的訊號傳遞過程，導致對於細胞移動造成影響。同時，此研究成果顯示PEP1在血管新生中扮演相當重要的角色，而經由更多有關分子機制的調查，將可在血管功能不全以及癌症上提供更深層的研究。

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.

1. Folkman, J. Angiogenesis: an organizing principle for drug discovery? Nature reviews 6, 273-286 (2007).
2. Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature medicine 1, 27-31 (1995).
3. Lamalice, L., Le Boeuf, F. & Huot, J. Endothelial cell migration during angiogenesis. Circulation research 100, 782-794 (2007).
4. Munoz-Chapuli, R., Quesada, A.R. & Angel Medina, M. Angiogenesis and signal transduction in endothelial cells. Cell Mol Life Sci 61, 2224-2243 (2004).
5. Sholley, M.M., Ferguson, G.P., Seibel, H.R., Montour, J.L. & Wilson, J.D. Mechanisms of neovascularization. Vascular sprouting can occur without proliferation of endothelial cells. Laboratory investigation; a journal of technical methods and pathology 51, 624-634 (1984).
6. Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Developmental biology 248, 307-318 (2002).
7. Childs, S., Chen, J.N., Garrity, D.M. & Fishman, M.C. Patterning of angiogenesis in the zebrafish embryo. Development (Cambridge, England) 129, 973-982 (2002).
8. Nakayama, M., Kikuno, R. & Ohara, O. Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs. Genome research 12, 1773-1784 (2002).
9. Kondo, A., et al. A new paxillin-binding protein, PAG3/Papalpha/KIAA0400, bearing an ADP-ribosylation factor GTPase-activating protein activity, is involved in paxillin recruitment to focal adhesions and cell migration. Molecular biology of the cell 11, 1315-1327 (2000).
10. Turner, C.E. Paxillin interactions. Journal of cell science 113 Pt 23, 4139-4140 (2000).
11. Brown, M.C. & Turner, C.E. Paxillin: adapting to change. Physiological reviews 84, 1315-1339 (2004).
12. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E.L. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of molecular biology 305, 567-580 (2001).
13. Sigrist, C.J., et al. PROSITE: a documented database using patterns and profiles as motif descriptors. Briefings in bioinformatics 3, 265-274 (2002).
14. Letunic, I., et al. SMART 4.0: towards genomic data integration. Nucleic acids research 32, D142-144 (2004).
15. Corpet, F. Multiple sequence alignment with hierarchical clustering. Nucleic acids research 16, 10881-10890 (1988).
16. Tatusova, T.A. & Madden, T.L. BLAST 2 Sequences, a new tool for comparing protein and nucleotide sequences. FEMS microbiology letters 174, 247-250 (1999).
17. Maruyama, Y. The human endothelial cell in tissue culture. Z Zellforsch Mikrosk Anat 60, 69-79 (1963).
18. Jaffe, E.A., Nachman, R.L., Becker, C.G. & Minick, C.R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. The Journal of clinical investigation 52, 2745-2756 (1973).
19. Teplizki, H., et al. ELISA measurement of antibody titer to purified protein derivative and mycobacteria-derived phosphoglycolipids: tool for diagnosing active pulmonary tuberculosis. Israel journal of medical sciences 23, 1121-1124 (1987).
20. Scotti, P.D. End-point dilution and plaque assay methods for titration of cricket paralysis virus in cultured Drosophila cells. The Journal of general virology 35, 393-396 (1977).
21. Nielsen, L.K., Smyth, G.K. & Greenfield, P.F. Accuracy of the endpoint assay for virus titration. Cytotechnology 8, 231-236 (1992).
22. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72, 248-254 (1976).
23. Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 (1970).
24. Dou, L., et al. The uremic solutes p-cresol and indoxyl sulfate inhibit endothelial proliferation and wound repair. Kidney international 65, 442-451 (2004).
25. Falk, W., Goodwin, R.H., Jr. & Leonard, E.J. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. Journal of immunological methods 33, 239-247 (1980).
26. Higa, F., et al. Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila. Journal of clinical microbiology 36, 1392-1398 (1998).
27. Adams, J.C. & Lawler, J. The thrombospondins. The international journal of biochemistry & cell biology 36, 961-968 (2004).
28. Sipes, J.M., Krutzsch, H.C., Lawler, J. & Roberts, D.D. Cooperation between thrombospondin-1 type 1 repeat peptides and alpha(v)beta(3) integrin ligands to promote melanoma cell spreading and focal adhesion kinase phosphorylation. The Journal of biological chemistry 274, 22755-22762 (1999).
29. Armstrong, L.C. & Bornstein, P. Thrombospondins 1 and 2 function as inhibitors of angiogenesis. Matrix Biol 22, 63-71 (2003).
30. Roberts, D.D. Regulation of tumor growth and metastasis by thrombospondin-1. Faseb J 10, 1183-1191 (1996).
31. Ren, B., Yee, K.O., Lawler, J. & Khosravi-Far, R. Regulation of tumor angiogenesis by thrombospondin-1. Biochimica et biophysica acta 1765, 178-188 (2006).
32. Ruoslahti, E. & Pierschbacher, M.D. Arg-Gly-Asp: a versatile cell recognition signal. Cell 44, 517-518 (1986).
33. Ferrara, N. & Davis-Smyth, T. The biology of vascular endothelial growth factor. Endocrine reviews 18, 4-25 (1997).
34. Nishiya, N., Kiosses, W.B., Han, J. & Ginsberg, M.H. An alpha4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells. Nature cell biology 7, 343-352 (2005).
35. Uchida, H., Kondo, A., Yoshimura, Y., Mazaki, Y. & Sabe, H. PAG3/Papalpha/KIAA0400, a GTPase-activating protein for ADP-ribosylation factor (ARF), regulates ARF6 in Fcgamma receptor-mediated phagocytosis of macrophages. The Journal of experimental medicine 193, 955-966 (2001).
36. Hashimoto, S., et al. A novel mode of action of an ArfGAP, AMAP2/PAG3/Papa lpha, in Arf6 function. The Journal of biological chemistry 279, 37677-37684 (2004).
37. Horwitz, R. & Webb, D. Cell migration. Curr Biol 13, R756-759 (2003).
38. Ridley, A.J., et al. Cell migration: integrating signals from front to back. Science (New York, N.Y 302, 1704-1709 (2003).
39. Gastl, G., et al. Angiogenesis as a target for tumor treatment. Oncology 54, 177-184 (1997).
40. Cines, D.B., et al. Endothelial cells in physiology and in the pathophysiology of vascular disorders. Blood 91, 3527-3561 (1998).
41. Aird, W.C. Endothelial cell heterogeneity. Critical care medicine 31, S221-230 (2003).