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研究生：	陳雅惠 Chen, Ya-Huey
論文名稱：	探討angiostatin所調控的血管新生作用之分子機轉 Delineation of the molecular mechanism of angiogenesis mediated by angiostatin
指導教授：	吳梨華 Wu, Li-Wha
學位類別：	博士 Doctor
系所名稱：	醫學院 - 基礎醫學研究所 Institute of Basic Medical Sciences
論文出版年：	2007
畢業學年度：	95
語文別：	英文
論文頁數：	107
中文關鍵詞：	血管新生
外文關鍵詞：	angiostatin
相關次數：	點閱：70 下載：1
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Angiostatin於1994年時，在帶有惡性之Lewis Lung Carcinoma的老鼠之尿液中被發現，並進一步被確認其可以經由抑制血管內皮細胞之生長，移動與促進血管內皮細胞產生細胞凋亡，而達到抑制血管新生的作用。已知Angiostatin包含了有plasminogen中kringle 1到4的部分(K1-4)，但是近年來的研究報告指出，與angiostatin相似的分子如：kringle 1到3 (K1-3)與kringle 1到4.5 (K1-4.5)的分子，亦具有抑制血管新生的能力，甚至其抑制作用強於K1-4，然而對於angiostatin (K1-4)與angiostatin相似分子K1-3和K1-4.5對於抑制血管新生的有效性與作用機轉到目前為止並沒有清楚的報導與研究，所以首先我們利用廠商純化具有活性之angiostatin(K1-4)，來探討其抑制血管新生的分子作用機轉，進一步利用重組與複製缺陷的adenovirus攜帶K1-3，K1-4與K1-4.5的cDNA，進而感染宿主細胞，感染後可以在細胞內產製K1-3，K1-4與K1-4.5蛋白，同時這些蛋白可以被分泌到細胞外的培養液中，透過收集這些細胞之培養液，進一步將其作用於血管內皮細胞，或者以直接感染血管內皮細胞後，使得K1-3，K1-4與K1-4.5蛋白可於血管內皮細胞中產製，並進而直接作用於血管內皮細胞的方式，來研究探討這些不同型之angiostatin的抑制效用與分子作用機制。目前我們發現這些不同型之angiostatin對於抑制血管內皮細胞之增生、移動、形成管狀結構、或促進血管內皮細胞產生凋亡的能力各有不同，但其分子作用之機轉大約相似。此不同型之angiostatin可以透過誘導p53、FasL蛋白之表現，及降低Akt激酶活性，並且使基因的表現失控，進而執行其抑制血管新生作用的工作，同時我們亦發現此不同型之angiostatin執行此抑制血管新生作用是需要透過angiostatin之接受器，integrin-αvβ3與ATPase synthase的協助，來完成此抑制工作。
最透過互補DNA之微矩陣基因片的實驗(cDNA mciroarray assay)，我們進一步的發現angiostatin(K1-4)相似分子K1-3和K1-4.5可以使E-selectin在mRNA上的表現量顯著的增加，尤其以K1-3更為明顯。E-selectin在血管內皮細胞中是一個重要的可被誘導的血球細胞黏著到血管內皮細胞上的貼附蛋白，而在其它的細胞中，其亦參與在細胞與細胞黏著吸附作用上，同時文獻也指出其與血管新生作用有相關性。其它之血管新生作用之抑制因子，如AGM-1470 (TNP-470)與K1-3皆可使得E-selectin在mRNA層面之表現量上升，且另一個血管新生抑制因子endostatin其執行抑制血管新生作用是需要E-selectin的存在，因此總結上述的結果，我們進一步探討E-selectin的表現是如何受到K1-3的調控及其在K1-3抑制血管新生的角色。由我們的實驗中發現內皮細胞內的E-selectin大量表現，是經由活化AP1與Ets-1轉錄因子，這些轉錄因子可以直接作用於E-selectin之啟動子，使E-selectin的mRNA表現增加進而轉錄形成蛋白，被K1-3誘導而表現的E-selectin可以進一步的移動到內皮細胞膜的lipid raft，與其它的蛋白或激酶作用，同時亦可使得JNK激酶的磷酸化增加，此磷酸化增加可以正向的使的AP1轉錄因子活性增加同時也可能讓細胞對存在有血管抑制因子產生反應。總合之，E-selectin是angiostatin於進行抗血管新生作用中，一個新的作用標的蛋白。

Angiostatin, a circulating inhibitor of angiogenesis, is an internal fragment of plasminogen that contains the first four kringle domains (K1-4) of plasminogen. Further studies showed that angiostatin-like molecules consisting of only the first three kringle domains (K1-3) was more antiangiogenic than K1-4, and that there is the existence of a naturally occurring isoform, K1-4.5. It is not clear whether different isoforms of angiostatin utilized similar or distinct pathways to mediate anti-angiogenesis. We first examined the signaling pathway mediated by proapoptotic angiostatin, K1-4. Inductions of p53-mediated intrinsic and FasL-mediated extrinsic death signaling pathways are involved in anti-angiogenic action of K1-4. We then compared the signaling pathways and mRNA expression profiles modulated by K1-3, K1-4, and K1-4.5. Although the extent of anti-angiogenic potency might vary among different isoforms, K1-3 or K1-4.5 shared similar death pathways with those mediated by K1-4. Moreover, all three forms of angiostatin induced a similar subset of mRNA expression with some variations. The common pathways shared by K1-3, K1-4, and K1-4.5 might be used as new therapeutic targets for anti-angiogenic therapy. Among the deregulated genes, the expression of E-selectin, an adhesion molecule, was not only induced by all isoforms but also previously implicated in antiangiogenic action of endostatin. Since K1-3 had the highest ability to induce E-selectin, we then used K1-3 to study the underlying mechanism responsible for the K1-3-induced expression of E-selectin. RT-PCR and western blotting analyses confirmed the time-dependent increase of E-selectin mRNA and protein induced by K1-3. Moreover, subcellular fraction and immunofluorescence microscopy confirmed the predominant presence of K1-3-induced E-selectin in lipid raft. Promoter driven reporter assays demonstrated both AP1 and Ets-1 binding sites on the E-selectin promoter were crucial for the induction of E-selectin. EMSA and ChIp assays confirmed the in vitro and in vivo binding of AP1 complex and Ets-1. Repression of JNK, a N-terminal kinase for c-Jun, significantly suppressed the K1-3 induced expression of E-selectin, suggesting a requirement of JNK activation for induction of E-selectin. The positive involvement of E-selectin in the anti-angiogenic action of K1-3 was, respectively, confirmed by overexpression and knockdown of E-selectin. In summary, we have identified the E-selectin as a novel target for the antiangiogenic action of angiostatin.

Contents -------------------------------------------------------------------------------------- i
Figure contents ----------------------------------------------------------------------------- v
Abbreviation --------------------------------------------------------------------------------- viii
中文摘要 ------------------------------------------------------------------------------------ 1
Abstract --------------------------------------------------------------------------------------- 3
Chapter 1 Introduction ----------------------------------------------------------------- 5
1-1 Blood vessels formation ---------------------------------------------------------- 5
1-2 Vasculogenesis --------------------------------------------------------------------- 5
1-3 Angiogenesis ----------------------------------------------------------------------- 6
1-4 The angiogenic switch ------------------------------------------------------------ 6
1-5 Tumor neovascularization -------------------------------------------------------- 7
1-6 Structure and function of tumor vessels ---------------------------------------- 7
1-7 Angiogenesis in non-neoplastic disease ---------------------------------------- 8
1-8 Tumor dormancy ------------------------------------------------------------------ 9
1-9 The discovery of agniostatin ----------------------------------------------------- 10
1-10 The structure of angiostatin---------------------------------------------------- 11
1-11 Individual kringle domains and angiogenesis inhibition ------------------ 12
1-12 Production of multiple forms of angiostatin -------------------------------- 12
1-13 The function of multiple forms of angiostatin ------------------------------ 13
1-14 The mechanisms whereby angiostatin execute anti-angiogenesis-------- 14
1-15 The role of E-selectin in modulation of anti-angiogenesis ---------------- 16
1-16 Signaling pathway mediated by E-selectin ---------------------------------- 16
1-17 The specific aim----------------------------------------------------------------- 18
Chapter 2 Materials and methods ------------------------------------------------------ 19
2-1 Materials ---------------------------------------------------------------------------- 19 
2-2 Antibody lists ---------------------------------------------------------------------- 21
2-3 Cell culture ------------------------------------------------------------------------- 22
2-4 Cell proliferation assay ----------------------------------------------------------- 22
2-5 Apoptosis assay by flow cytometry --------------------------------------------- 22
2-6 Western blotting analysis --------------------------------------------------------- 23
2-7 RT-PCR followed by Southern blot hybridization ---------------------------- 23
2-8 Subcellular distribution of cytochrome c -------------------------------------- 24
2-9 Caspase 3 activity assay --------------------------------------------------------- 24
2-10 Establishment and titration of recombinant adenovirus ------------------- 24
2-11 Collection of conditioned media (CM) following adenoviral infection of 549 cells ------------------------------------------------------------------------------- 25
2-12 Immunoblotting of K1-3, K1-4, and K1-4.5 in the CM ------------------- 25
2-13 Cell proliferation assay with CM --------------------------------------------- 26
2-14 Cell migration ------------------------------------------------------------------- 26
2-15 Tube formation assay ---------------------------------------------------------- 26
2-16 Apoptosis assay by flow cytometry with CM ------------------------------ 27
2-17 Western blot analysis with CM ----------------------------------------------- 27
2-18 RT-PCR and Southern blot analysis ----------------------------------------- 27
2-19 cDNA microarray analysis ---------------------------------------------------- 28
2-20 HL-60 cell adhesion assay ---------------------------------------------------- 28
2-21 Transfection with lipofetamine 2000 and reporter gene assay ----------- 29
2-22 Lipid raft isolation assay ------------------------------------------------------ 29
2-23 Immunoflorescence assay ----------------------------------------------------- 30
2-24 Chromatin immunoprecipitation assay -------------------------------------- 30
2-25 Preparation of nuclear extracts ----------------------------------------------- 31
2-26 Gel electrophoretic mobility shift assays (EMSA) ------------------------ 31
2-27 SiRNA of E-selectin ---------------------------------------------------------- 32
2-28 Statistical analysis ------------------------------------------------------------- 32
Chapter 3 Results -------------------------------------------------------------------------- 33
3-1 Angiostatin inhibits the proliferation while increasing apoptosis of HUVEC serum-deprived ------------------------------------------------------------------- 33
3-2 Angiostatin increases the levels of p53 and Bax ----------------------------- 33
3-3 Angiostatin induces the release of cytochrome c into the cytosol possibly via cleavage of Bid ------------------------------------------------------------------- 34
3-4 Angiostatin promotes the activation of caspase 3 --------------------------- 35
3-5 Angiostatin both inhibited cell proliferation and induced apoptosis ------ 35
3-6 Protein levels and functions of K1-3, K1-4 and K1-4.5 in CM ------------ 36
3-7 K1-3, K1-4 or K1-4.5 induce the expression of p53, Bax, p21, and DAPK-1--------------------------------------------------------------------------- 37
3-8 Participation of FasL, c-FLIP, and caspase 8 in antiangiogenesis mediated by K1-3, K1-4 and K1-4.5 ---------------------------------------------------------- 37
3-9 Neutralization of FasL attenuated the ability of K1-3, K1-4 and K1-4.5 to induce apoptosis ----------------------------------------------------------------- 38
3-10 Attenuation of VEGF-induced phosphorylation of AKT by K1-3, K1-4 and K1-4.5 ---------------------------------------------------------------------------- 38
3-11 Decreased angiostatin-mediated apoptosis by anti- α-ATPase or anti-integrin αvβ3 antibodies ----------------------------------------------- 39
3-12 Differential regulation of gene expression mediated by K1-3, K1-4 and K1-4.5 -------------------------------------------------------------------------- 39
3-13 Increased HL-60 cell adhesion to endothelial cells by K1-3, K1-4 or K1-4.5
------------------------------------------------------------------------------------ 40
3-14 Three forms of angiostatin (K1-3, K1-4, and K1-4.5) share similar pathway
to block angiogenesis -------------------------------------------------------- 41
3-15 The expression of E-selectin in HUVEC mediated by multiple forms of angiostatin in RNA and protein level ------------------------------------- 41
3-16 The promoter activity of E-selectin in HUVEC regulated by multiple forms of angiostatin ------------------------------------------------------------------- 42
3-17 The activation of E-selectin promoter mediated by K1-3 required the AP-1 and Ets-1 ------------------------------------------------------------------------ 43
3-18 The expression of AP-1 and Ets-1 and the potential of binding to E-selectin promoter ----------------------------------------------------------------------- 44
3-19 The localization of E-selectin in lipid raft in HUVEC mediated by K1-3
------------------------------------------------------------------------------------- 45
3-20 The signaling pathways involved in the K1-3-induced expression of E-selectin ----------------------------------------------------------------------- 45
3-21 Effects of overexpression or knockdown of E-selectin in the K1-3-mediated endothelial cell proliferation -------------------------------------------------- 46
3-22 The K1-3 increased the expression of E-selectin and then localized to lipid raft to increase the phosphorylation of JNK ------------------------------- 47
Chapter 4 Discussions -------------------------------------------------------------------- 48
References ----------------------------------------------------------------------------------- 56
Figures --------------------------------------------------------------------------------------- 66
Tables ---------------------------------------------------------------------------------------- 98
Appendix ----------------------------------------------------------------------------------- 99
Publication --------------------------------------------------------------------------------- 107
Figure contents
Fig. 1 Inbition of HUVEC proliferation and increased apoptosis by angiostatin.
------------------------------------------------------------------------------------ 66
Fig. 2 Deregulation of p53 and Bax by angiostatin. ------------------------------ 67
Fig. 3 Release of cytochrome c into cytosol induced by angiostatin and increased cleavage of Bid by angiostatin. ----------------------------------------------- 68
Fig. 4 Increased FasL mRNA and activation of caspase 3 but decreased c-Flip mRNA by angiostatin. ---------------------------------------------------------- 69
Fig. 5 Angiostatin both inhibited cell proliferation and induced apoptosis. ---- 70
Fig. 6 K1-3, K1-4 or K1-4.5 inhibits proliferation, migration, and tube formation and induce apoptosis. ----------------------------------------------------------- 71
Fig. 7 K1-3, K1-4 or K1-4.5 enhances the expression of p53, p21, Bax and DAPK1. -------------------------------------------------------------------------- 73
Fig. 8 K1-3, K1-4 or K1-4.5 increases the level of FasL protein and cleavage of caspase 8 while reducing cFLIP mRNA expression. ---------------------- 74
Fig. 9 Neutralization of Fas attenuates the pro-apoptotic ability of angiostatin molecules to different extents ------------------------------------------------ 75
Fig. 10 K1-3, K1-4 or K1-4.5 attenuates the activity of AKT. ------------------- 76
Fig. 11 KI-3, KI-4 or KI-4.5 induces apoptosis in endothelial cells through both integrin-avβ3 and ATP synthase. ------------------------------------------ 77
Fig. 12 Gene deregulation mediated by K1-3, K1-4 or K1-4.5. ----------------- 78
Fig. 13 RT-PCR confrimed the gene deregulation mediated by K1-3, K1-4 or K1-4.5. ------------------------------------------------------------------------- 79
Fig. 14 Three forms of angiostatin, K1-3, K1-4 and K1-4.5, share similar pathways to inhibit angiogenesis. ------------------------------------------------------ 80
Fig. 15 E-selectin expression in HUVEC activated by K1-3 at different time course. 
----------------------------------------------------------------------------------- 81
Fig. 16 The expression of E-selectin in RNA and protein levels. -------------- 82
Fig. 17 Activation of the E-selectin promoter by angiostatin. ------------------ 83
Fig. 18 Scheme diagram depicting E-selectin promoter construct used in the experiments. ------------------------------------------------------------------ 84
Fig. 19 AP1 (c-Jun) and Ets-1 are required for the induction of E-selectin promoter. ---------------------------------------------------------------------- 85
Fig. 20 Overexpression of AP-1 and Ets-1 stimulated the E-selectin promoter activity. ------------------------------------------------------------------------ 86
Fig. 21 AP-1(c-Jun) and Ets-1 activated E-selectin promoter. ------------------ 87
Fig. 22 The mRNA expression of c-Jun, c-Fos, and Ets-1 in HUVEC activated by K1-3 at different time points. ---------------------------------------------- 88
Fig. 23 The protein expression of E-selectin, AP-1 (c-Jun) and Ets-1 in HUVEC treated with K1-3. ------------------------------------------------------------ 89
Fig. 24 Specificity of the AP1 and Ets-1 binding. -------------------------------- 90
Fig. 25 AP-1 (c-Jun) and Ets-1 binding to E-selectin promoter in vivo by using ChIp assay method. ----------------------------------------------------------------- 91
Fig. 26 Lipid raft fractionation and IF of E-selectin in HUVECs mediated by K1-3.
----------------------------------------------------------------------------------- 92
Fig. 27 The phosphorylation of MEK family in HUVEC treated with K1-3.
---------------------------------------------------------------------------------- 93
Fig. 28 The expression of E-selectin in presence of kinase inhibitor. --------- 94
Fig. 29 siRNA silencing of exogenous E-selectin gene expression in 293 cells and HUVECs and endogenous in HUVEC. ---------------------------------- 95
Fig. 30 The proliferation assay of HUVEC while overexpressed or knockdown the E-selectin. ------------------------------------------------------------------- 96
Fig. 31 The K1-3 increased the expression of E-selectin and then localized to lipid raft to increase the phosphorylation of JNK. ---------------------------- 97
                                    

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