臺灣博碩士論文加值系統

血管網絡的形成是一個被高度調控的過程。在此之中，包含了對血管內皮細胞移動、分支、和連接精密的調控。根據先前的文獻，內皮細胞正確的方向性移動和分支決定於正常的內皮細胞突出結構。然而方向性突出結構的相關機制仍未明確。在此篇文章中，我們提出一個新穎蛋白質—凝血酶敏感蛋白區域包含7A，簡稱THSD7A。這個新穎蛋白表現在班馬魚發育中的神經系統，而我們認為這個新穎蛋白具有調控內皮細胞突出結構的功能。
從我們的研究結果顯示，當我們利用morpholino抑制THSD7A蛋白質的表現時，受精30小時的班馬魚體節間血管的生長受到干擾。為了得到更多研究資料，我們利用共軛焦顯微鏡觀察THSD7A抑制的班馬魚，並檢察其體節間血的內皮細胞是否表現特殊形態和行為。結果顯示，當THSD7A被抑制時，血管內皮細胞被錯誤的引導並停留在細胞移動的初期，細胞突出結構廣泛的被延伸呈現扇形，因此導致體節間血管的生長被延遲。這個形態暗示內皮層細胞在縮回突出結構的機制上出了問題。除此之外，體節間血管上的內皮細胞也呈現不正常的分叉和錯誤連接的現像，使得受精後34小時班馬魚的血管網絡出現過度雜亂的圖形。
另一方面，一種在生物體外的細胞實驗—三度空間膠原蛋白的血管新生測試，也被運用來探討THSD7A的特殊功能。從此模式得到的研究結果顯示，THSD7A確實跟血管新生所產生的網絡圖形有關，包括調控內皮細胞的突出物結構和連接。統計資料也暗示我們THSD7A可能扮演了雙重性功能的角色，在不同濃度下，此蛋白對血管新生有不一樣的影響。
總而言之，這篇論文提供了THSD7A這個新穎蛋白影響血管新生的可能性，THSD7A可能藉由調控內皮細胞突出物，控制內皮細胞的分支和連接，進而影響血管新生的網絡圖形。

Vascular patterning is a highly organized process in angiogenesis. It involves precise control of endothelial cell (EC) migration, branching, and connection. Based on previous studies, directed EC migration and branching depends on correct protrusions of ECs. However, the underlying mechanism of directional EC protrusions is still unclear. Here we propose a novel secreted protein, THSD7A, which is expressed in the zebrafish developing neuronal system, contributes to EC protrusion regulation.
From our data, knockdown of THSD7A expression by injecting morpholino disrupted intersegmental vessel (ISV) sprouting of zebrafish at 30 hour-post-fertilization (hpf) stage. To acquire more information, we further examined the behavior and morphology of endothelial tip cells on the ISVs of Thsd7a-knockdown zebrafish by confocal microscopy. The data showed that morpholino knockdown of THSD7A slowed down ISV growth by misleading endothelia cells to stay longer in early stage of dynamic protrusion process, in which ECs extended numerous protrusions like a fan-shape. This phenotype implied malfunction of retracting excessive EC protrusions. Besides, ECs of ISVs formed ectopic EC branching and false connection, resulting in complex ISV pattern in 34hpf zebrafish.
On the other hand, an in vitro system, 3D collagen angiogenesis assay, was constructed for investigating the specific role of THSD7A. The data from in vitro 3D collagen angiogenesis assay proved that THSD7A was related to angiogenic patterning, including EC protrusions and connections. Statistic analysis also implied that THSD7A played a dual role in angiogenesis depending on its concentration.
Taken the in vivo and in vitro data together, this work suggests that THSD7A may mediate EC protrusion during angiogenic sprouting and plays an essential role in angiogenic patterning as a regular of EC branching and connection.

摘要 I
Abstract II
List of contents III
List of figures IV
1 Introduction 1
2 Materials and Methods 3
2.1 Fish stocks 3
2.2 Morpholino microinjection 3
2.3 Zebrafish THSD7A cloning and rescue 3
2.4 Real Time-quantitative PCR analysis 3
2.5 HUVEC isolation 4
2.6 Cell cultures of HUVEC, HEK 293 T 4
2.7 HEK 293 T expression system 5
2.8 3D collagen angiogenesis assay 5
2.9 ELISA (Enzyme-Linked Immunosorbent Assay) 6
3 Results and Discussions 8
3.1 THSD7A is required for ISV angiogenesis during zebrafish development 8
3.2 ISVs stayed in early step of migration process displaying protrusions without major direction in THSD7A-knockdown zebrafish at 29~34 hpf stage. 9
3.3 ISVs showed abnormal protrusions and connections across somite in THSD7A-knockdown zebrafish at 34 hpf stage. 10
3.4 Decreasing Flk1(VEGFR2) and increasing Flt4 (VEGFR3) expression in THSD7A-knockdown zebrafish suggested a possible genetic interaction. 11
3.5 Recombinant THSD7A mediated HUVEC angiogenic patterning in 3D collagen angiogenesis assay. 12
3.6 Recombinant THSD7A mediated EC protrusions in 3D collagen angiogenesis assay. 14
4 References 16

1. Folkman, J. & D'Amore, P.A. Blood vessel formation: what is its molecular basis? Cell 87, 1153-1155 (1996).
2. Patan, S. Vasculogenesis and angiogenesis as mechanisms of vascular network formation, growth and remodeling. J Neurooncol 50, 1-15 (2000).
3. Ausprunk, D.H. & Folkman, J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc Res 14, 53-65 (1977).
4. Marin-Padilla, M. Early vascularization of the embryonic cerebral cortex: Golgi and electron microscopic studies. J Comp Neurol 241, 237-249 (1985).
5. Fischer, R.S., Gardel, M., Ma, X., Adelstein, R.S. & Waterman, C.M. Local cortical tension by myosin II guides 3D endothelial cell branching. Curr Biol 19, 260-265 (2009).
6. Klosovskii, B.N. & Zhukova, T.P. [Effect of colchicine on remote phases of growing capillaries in the brain.]. Arkh Patol 35(3), 38-44 (1963).
7. Gerhardt, H., et al. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161, 1163-1177 (2003).
8. Ruhrberg, C., et al. Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis. Genes Dev 16, 2684-2698 (2002).
9. Huber, A.B., Kolodkin, A.L., Ginty, D.D. & Cloutier, J.F. Signaling at the growth cone: ligand-receptor complexes and the control of axon growth and guidance. Annu Rev Neurosci 26, 509-563 (2003).
10. Covassin, L.D., Villefranc, J.A., Kacergis, M.C., Weinstein, B.M. & Lawson, N.D. Distinct genetic interactions between multiple Vegf receptors are required for development of different blood vessel types in zebrafish. Proc Natl Acad Sci U S A 103, 6554-6559 (2006).
11. Dameron, K.M., Volpert, O.V., Tainsky, M.A. & Bouck, N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265, 1582-1584 (1994).
12. Guo, N., Krutzsch, H.C., Inman, J.K. & Roberts, D.D. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res 57, 1735-1742 (1997).
13. Hsu, S.C., et al. Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res 56, 5684-5691 (1996).
14. Asch, A.S., Barnwell, J., Silverstein, R.L. & Nachman, R.L. Isolation of the thrombospondin membrane receptor. J Clin Invest 79, 1054-1061 (1987).
15. Li, W.X., Howard, R.J. & Leung, L.L. Identification of SVTCG in thrombospondin as the conformation-dependent, high affinity binding site for its receptor, CD36. J Biol Chem 268, 16179-16184 (1993).
16. Frieda, S., Pearce, A., Wu, J. & Silverstein, R.L. Recombinant GST/CD36 fusion proteins define a thrombospondin binding domain. Evidence for a single calcium-dependent binding site on CD36. J Biol Chem 270, 2981-2986 (1995).
17. Greenwalt, D.E., et al. Membrane glycoprotein CD36: a review of its roles in adherence, signal transduction, and transfusion medicine. Blood 80, 1105-1115 (1992).
18. Swerlick, R.A., Lee, K.H., Wick, T.M. & Lawley, T.J. Human dermal microvascular endothelial but not human umbilical vein endothelial cells express CD36 in vivo and in vitro. J Immunol 148, 78-83 (1992).
19. Petzelbauer, P., Bender, J.R., Wilson, J. & Pober, J.S. Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture. J Immunol 151, 5062-5072 (1993).
20. Giancotti, F.G. Complexity and specificity of integrin signalling. Nat Cell Biol 2, E13-14 (2000).
21. Klemke, R.L., et al. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 137, 481-492 (1997).
22. Davis, G.E., Black, S.M. & Bayless, K.J. Capillary morphogenesis during human endothelial cell invasion of three-dimensional collagen matrices. In Vitro Cell Dev Biol Anim 36, 513-519 (2000).
23. Senger, D.R., et al. The alpha(1)beta(1) and alpha(2)beta(1) integrins provide critical support for vascular endothelial growth factor signaling, endothelial cell migration, and tumor angiogenesis. Am J Pathol 160, 195-204 (2002).
24. Lawson, N.D. & Weinstein, B.M. Arteries and veins: making a difference with zebrafish. Nat Rev Genet 3, 674-682 (2002).
25. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B. & Schilling, T.F. Stages of embryonic development of the zebrafish. Dev Dyn 203, 253-310 (1995).
26. Maruyama, Y. The human endothelial cell in tissue culture. Z Zellforsch Mikrosk Anat 60, 69-79 (1963).
27. 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. J Clin Invest 52, 2745-2756 (1973).
28. Heisenberg, C.P., et al. Silberblick/Wnt11 mediates convergent extension movements during zebrafish gastrulation. Nature 405, 76-81 (2000).
29. Leung, T., et al. Zebrafish G protein gamma2 is required for VEGF signaling during angiogenesis. Blood 108, 160-166 (2006).
30. Wolff, J.R. & Bar, T. 'Seamless' endothelia in brain capillaries during development of the rat's cerebral cortex. Brain Res 41, 17-24 (1972).
31. Lu, X., et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 432, 179-186 (2004).
32. Torres-Vazquez, J., et al. Semaphorin-plexin signaling guides patterning of the developing vasculature. Dev Cell 7, 117-123 (2004).
33. Small, J.V., Stradal, T., Vignal, E. & Rottner, K. The lamellipodium: where motility begins. Trends Cell Biol 12, 112-120 (2002).
34. Pollard, S.M., et al. Essential and overlapping roles for laminin alpha chains in notochord and blood vessel formation. Dev Biol 289, 64-76 (2006).
35. Leslie, J.D., et al. Endothelial signalling by the Notch ligand Delta-like 4 restricts angiogenesis. Development 134, 839-844 (2007).
36. Bussmann, J., Bakkers, J. & Schulte-Merker, S. Early endocardial morphogenesis requires Scl/Tal1. PLoS Genet 3, e140 (2007).
37. Liao, W., et al. The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation. Development 124, 381-389 (1997).
38. Thompson, M.A., et al. The cloche and spadetail genes differentially affect hematopoiesis and vasculogenesis. Dev Biol 197, 248-269 (1998).
39. Siekmann, A.F. & Lawson, N.D. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445, 781-784 (2007).
40. Nasevicius, A., Larson, J. & Ekker, S.C. Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant. Yeast 17, 294-301 (2000).
41. Martyn, U. & Schulte-Merker, S. Zebrafish neuropilins are differentially expressed and interact with vascular endothelial growth factor during embryonic vascular development. Dev Dyn 231, 33-42 (2004).
42. Lawson, N.D., Mugford, J.W., Diamond, B.A. & Weinstein, B.M. phospholipase C gamma-1 is required downstream of vascular endothelial growth factor during arterial development. Genes Dev 17, 1346-1351 (2003).
43. Reynolds, A.R., et al. Elevated Flk1 (vascular endothelial growth factor receptor 2) signaling mediates enhanced angiogenesis in beta3-integrin-deficient mice. Cancer Res 64, 8643-8650 (2004).
44. Lohela, M., Saaristo, A., Veikkola, T. & Alitalo, K. Lymphangiogenic growth factors, receptors and therapies. Thromb Haemost 90, 167-184 (2003).
45. Hogan, B.M., et al. Vegfc/Flt4 signalling is suppressed by Dll4 in developing zebrafish intersegmental arteries. Development 136, 4001-4009 (2009).
46. Soga, N., Connolly, J.O., Chellaiah, M., Kawamura, J. & Hruska, K.A. Rac regulates vascular endothelial growth factor stimulated motility. Cell Commun Adhes 8, 1-13 (2001).
47. Lamalice, L., Houle, F., Jourdan, G. & Huot, J. Phosphorylation of tyrosine 1214 on VEGFR2 is required for VEGF-induced activation of Cdc42 upstream of SAPK2/p38. Oncogene 23, 434-445 (2004).
48. Ispanovic, E., Serio, D. & Haas, T.L. Cdc42 and RhoA have opposing roles in regulating membrane type 1-matrix metalloproteinase localization and matrix metalloproteinase-2 activation. Am J Physiol Cell Physiol 295, C600-610 (2008).
49. Pollard, T.D. & Borisy, G.G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453-465 (2003).
50. Kendall, R.L. & Thomas, K.A. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci U S A 90, 10705-10709 (1993).
51. Sawano, A., Takahashi, T., Yamaguchi, S., Aonuma, M. & Shibuya, M. Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factor, which is related to vascular endothelial growth factor. Cell Growth Differ 7, 213-221 (1996).
52. Strongin, A.Y., et al. Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. J Biol Chem 270, 5331-5338 (1995).
53. Bernardo, M.M. & Fridman, R. TIMP-2 (tissue inhibitor of metalloproteinase-2) regulates MMP-2 (matrix metalloproteinase-2) activity in the extracellular environment after pro-MMP-2 activation by MT1 (membrane type 1)-MMP. Biochem J 374, 739-745 (2003).