臺灣博碩士論文加值系統

During atherogenesis, inflammation and coagulation play an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties, it can also regulate cell migration and angiogenesis. Transcription factor, KLF2, has been shown to participate in the regulation of the expression of eNOS, TM and other anti-inflammation genes that can regulate the physiological function of endothelial cells (ECs). ECs are constantly under the influence of flow-induced shear stress. Therefore, shear stress is an important regulator of EC functions. In this study, we focused on the signaling pathway of TM and KLF2 under the shear stress stimulation and the regulatory mechanism of TM protein stability.

Shear stress elevated TM promoter activity in ECs after 6 hours under both high (25 dyn/cm2) and low (5 dyn/cm2) shear stress conditions, but high shear had more eminent induction (12 folds vs. unsheared control) than low shear (6 folds vs. control). The mRNA level of TM was increased more than 2 folds in comparison with unsheared control and so did the mRNA level of KLF2 (more than 100-fold increase). As for TM protein level, there was a significant difference between high shear (60% increase) and low shear (23% increase) conditions, but no difference in KLF2 protein level was observed under different shear conditions. These results suggest that shear stress increases the protein stability of TM and the effect is proportional to the magnitude of shear stress.

We further explored the regulatory mechanism of TM stability. To simulate the condition of shear-induced NO release, ECs were treated with NOC18 (an NO donor), and we found that TM protein level increased 0.8 folds after 3 hours but no significant increase was observed after 5 hours. It is likely that NO may not be a major factor that regulates TM stability or there are too many down-stream signal molecules activated by NO to observe significant effect of NO on TM. Subsequently, ECs were treated with NAC to mimic the effect of antioxidants which were present in ECs exposed to high shear stress. The results showed that the TM protein level was almost totally down-regulated after 5 hours of shear treatment, but the soluble form of TM in the medium was increased. It suggests that NAC (or other antioxidants) may activate specific protease(s) to cleave TM into the solube form that can promote cell migration or angiogenesis.

To explore the effect of phosphorylation/dephosphorylation on the stability of TM, tyrosine kinase inhibitor (PP2) and PTP inhibitor (Na3VO4) were used. We found that TM protein level was increased about 2.2 folds in the presence of PP2, and decreased to 0.2 fold in the presence of Na3VO4 in ECs stimulated by VEGF for 5 hours. Based on these findings, we proposed that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. In order to verify this assumption, Y534D (superactive) and Y534A (dominant negative) TM mutants were transfected into HUVECs. We found that Y534D was more unstable in the presence of Na3VO4 but Y534A was not affected by PP2 or Na3VO4. Therefore the phosphorylation of Y534 may destabilize TM protein. Among various PTPs, we found that PTEN was involved in the regulatory mechanism of TM. PTEN silencing down-regulated TM protein level but had no influence on Y534A mutant, and this mechanism was found to be independent of PI3K/Akt pathway.

In summary, our data suggest that both high and low shear stresses up-regulate the TM promoter activity and KLF2 mRNA, but only high shear elevates the level of TM mRNA. Besides, shear stress dose-dependently increases the TM protein stability. Further studies indicate that NO may not play a significant role in the regulatory mechanism. As for NAC, it can increase the soluble form of TM, and this phenomenon is due to the cleavage of TM by some unknown protease(s). The key point in the regulation of TM stability seems to be the phosphorylation of Y534. Our results suggest that the phosphorylation of Y534, may be mediated by Src kinase family, decreases the stability of TM, but the activation of PTP such as PTEN enhances the TM protein stability.

目錄
誌謝 I
中文摘要 III
Abstract V
目錄 VII
圖目錄 XI
表目錄 XV
縮寫與符號說明 XVII
中英名詞對照 XXI
1. 緒論 1
1.1. 動脈粥狀硬化（Atherosclerosis） 1
1.2. 研究動機與目的 7
2. 文獻回顧 9
2.1. 血管內皮細胞與剪力 9
2.1.1. 血管內皮細胞 9
2.1.2. 剪力對血管內皮細胞之影響 13
2.1.3. 內皮細胞對於剪力之感測器 18
2.2. 凝血調節酶（Thrombomodulin, TM） 19
2.2.1. TM之結構與生理功能 19
2.2.2. TM對發炎機制之調控 25
2.2.3. TM對凝血機制之調控 26
2.2.4. TM對細胞附著之調控 28

2.2.5. TM對血管新生及細胞遷移之調控 29
2.2.6. TM表現量之調控 31
2.3. PTEN（Phosphatase and tensin homolog deleted on chromosome 10） 33
2.3.1. PTEN之結構 33
2.3.2. PTEN在生理功能上扮演的角色 36
2.4. Kruppel Like Factor 2(KLF2)之結構與生理功能 42
2.5. 一氧化氮（NO）對於內皮細胞之調控 45
3. 實驗藥品、儀器及方法 49
3.1. 實驗材料 49
3.1.1. 細胞培養及流動實驗所用材料 49
3.1.2. 實驗耗材 51
3.1.3. 細胞轉染所使用之材料 51
3.1.4. 西方墨點轉印法所用之材料 54
3.1.5. 同步定量聚合酶連鎖反應（Real-time quantitative PCR） 55
3.1.6. 啟動子活性測定法（Promoter Activity Assay） 56
3.2. 實驗儀器 57
3.3. 實驗原理與方法 59
3.3.1. 初級人類臍帶靜脈內細胞培養 59
3.3.2. 人類臍帶靜脈內皮細胞繼代培養於玻片 60
3.3.3. 人類臍帶靜脈內皮細胞繼代培養於培養皿 60
3.3.4. 牛動脈內皮細胞繼代陪養 61
3.3.5. 流動室之設計 61
3.3.6. 流動實驗之設計與流程 66
3.3.7. 全細胞之蛋白質（total cell lysate）的抽取 68
3.3.8. 蛋白質含量測定 68
3.3.9. 細胞內特定蛋白質含量測定Western Blot 69
3.3.10. 細胞內全RNA（total RNA）的收取 70
3.3.11. 細胞內特定mRNA含量測定：Real-Time quantitiative PCR 70
3.3.12. 以電穿孔（Electroporation）方式進行細胞轉染 72
3.3.13. 以Lipofectamine2000方式進行細胞轉染 73
3.3.14. 啟動子活性測定 73
3.3.15. β-Galactosidase assay..................................................................... 74
3.3.16. Luciferase assay.............................................................................. 74
4. 結果與討論............................................................................................................ 75
4.1. 不同剪力大小對於TM 及KLF2 之影響..................................................... 75
4.1.1. 不同剪力大小對TM 啟動子活性之調控.................................... 75
4.1.2. 不同剪力大小對TM 及KLF2 mRNA 之影響............................ 78
4.1.3. 不同剪力大小對TM 及KLF2 蛋白質之影響............................. 81
4.2. TM 蛋白質穩定性之量測.............................................................................. 84
4.3. 一氧化氮（NO）對於TM 穩定性之影響.................................................. 86
4.4. 細胞內氧化還原態對於TM 穩定性之影響................................................ 90
4.5. Src kinase family 與PTP 對TM 穩定性之調控........................................... 95
4.5.1. Src kinase family 對TM 穩定性之調控...................................... 95
4.5.2. 蛋白質酪胺酸去磷酸酶（PTP）對TM 穩定性之調控............. 98
4.6. TM 蛋白質穩定性受Y534 調控................................................................. 101
4.7. PTEN 對於調控內皮細胞TM 穩定性之影響............................................ 105
4.8. PI3K/Akt 路徑對TM 穩定性之影響........................................................... 109
4.9. Y534 決定TM 之蛋白質穩定性..................................................................111
4.10. 綜合討論.....................................................................................................114
5. 結論.......................................................................................................................119
5.1. 結論...............................................................................................................119
5.2. 未來研究方向.............................................................................................. 122
參考文獻...................................................................................................................... 123

Adams, T.E., and Huntington, J.A. (2006). Thrombin-cofactor interactions: structural insights into regulatory mechanisms. Arteriosclerosis, Thrombosis, and Vascular Biology 26, 1738-1745.
Anderson, K.P., Kern, C.B., Crable, S.C., and Lingrel, J.B. (1995). Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Kruppel-like factor: identification of a new multigene family. Molecular and Cellular Biology 15, 5957-5965.
Baker, S.J. (2007). PTEN enters the nuclear age. Cell 128, 25-28.
Behrendt, D., and Ganz, P. (2002). Endothelial function. From vascular biology to clinical applications. American Journal of Cardiology 90, 40L-48L.
Berliner, J.A., and Heinecke, J.W. (1996). The role of oxidized lipoproteins in atherogenesis. Free Radical Biology & Medicine 20, 707-727.
Berton, G., Mocsai, A., and Lowell, C.A. (2005). Src and Syk kinases: key regulators of phagocytic cell activation. Trends in Immunology 26, 208-214.
Bhattacharya, R., Senbanerjee, S., Lin, Z., Mir, S., Hamik, A., Wang, P., Mukherjee, P., Mukhopadhyay, D., and Jain, M.K. (2005). Inhibition of vascular permeability factor/vascular endothelial growth factor-mediated angiogenesis by the Kruppel-like factor KLF2. Journal of Biological Chemistry 280, 28848-28851.
Bieker, J.J. (2001). Kruppel-like factors: three fingers in many pies. Journal of Biological Chemistry 276, 34355-34358.
Bird, R., Stewart, W., and Lightfoot, E. (2002). Transport Phenomena. (New York, Wiley).
Bonner, J.C. (1994). Regulation of platelet-derived growth factor (PDGF) and alveolar macrophage-derived PDGF by alpha 2-macroglobulin. Annals of the New York Academy of Sciences 737, 324-338.


Botella, L.M., Sanchez-Elsner, T., Sanz-Rodriguez, F., Kojima, S., Shimada, J., Guerrero-Esteo, M., Cooreman, M.P., Ratziu, V., Langa, C., Vary, C.P., et al. (2002). Transcriptional activation of endoglin and transforming growth factor-beta signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury. Blood 100, 4001-4010.
Bourin, M.C., Lundgren-Akerlund, E., and Lindahl, U. (1990). Isolation and characterization of the glycosaminoglycan component of rabbit thrombomodulin proteoglycan. Journal of Biological Chemistry 265, 15424-15431.
Brach, M.A., Gruss, H.J., Riedel, D., Asano, Y., De Vos, S., and Herrmann, F. (1992). Effect of antiinflammatory agents on synthesis of MCP-1/JE transcripts by human blood monocytes. Molecular Pharmacology 42, 63-68.
Bradford, M.M. (1976). 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.
Bruemmer, D., Collins, A.R., Noh, G., Wang, W., Territo, M., Arias-Magallona, S., Fishbein, M.C., Blaschke, F., Kintscher, U., Graf, K., et al. (2003). Angiotensin II-accelerated atherosclerosis and aneurysm formation is attenuated in osteopontin-deficient mice. Journal of Clinical Investigation 112, 1318-1331.
Busse, R., and Fleming, I. (2003). Regulation of endothelium-derived vasoactive autacoid production by hemodynamic forces. Trends in Pharmacological Sciences 24, 24-29.
Calnek, D.S., and Grinnell, B.W. (1998). Thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor. Experimental Cell Research 238, 294-298.
Chien, S., Li, S., and Shyy, Y.J. (1998). Effects of mechanical forces on signal transduction and gene expression in endothelial cells. Hypertension 31, 162-169.
Collins, R.G., Velji, R., Guevara, N.V., Hicks, M.J., Chan, L., and Beaudet, A.L. (2000). P-Selectin or intercellular adhesion molecule (ICAM)-1 deficiency substantially protects against atherosclerosis in apolipoprotein E-deficient mice. Journal of Experimental Medicine 191, 189-194.
Conway, E.M., Liu, L., Nowakowski, B., Steiner-Mosonyi, M., and Jackman, R.W. (1994). Heat shock of vascular endothelial cells induces an up-regulatory transcriptional response of the thrombomodulin gene that is delayed in onset and does not attenuate. Journal of Biological Chemistry 269, 22804-22810.


Conway, E.M., Pollefeyt, S., Collen, D., and Steiner-Mosonyi, M. (1997). The amino terminal lectin-like domain of thrombomodulin is required for constitutive endocytosis. Blood 89, 652-661.
Conway, E.M., Pollefeyt, S., Cornelissen, J., DeBaere, I., Steiner-Mosonyi, M., Weitz, J.I., Weiler-Guettler, H., Carmeliet, P., and Collen, D. (1999). Structure-function analyses of thrombomodulin by gene-targeting in mice: the cytoplasmic domain is not required for normal fetal development. Blood 93, 3442-3450.
Conway, E.M., Van de Wouwer, M., Pollefeyt, S., Jurk, K., Van Aken, H., De Vriese, A., Weitz, J.I., Weiler, H., Hellings, P.W., Schaeffer, P., et al. (2002). The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. Journal of Experimental Medicine 196, 565-577.
Cully, M., You, H., Levine, A.J., and Mak, T.W. (2006). Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nat Rev Cancer 6, 184-192.
Dang, D.T., Pevsner, J., and Yang, V.W. (2000). The biology of the mammalian Kruppel-like family of transcription factors. International Journal of Biochemistry & Cell Biology 32, 1103-1121.
Davies, P.F., Shi, C., Depaola, N., Helmke, B.P., and Polacek, D.C. (2001). Hemodynamics and the focal origin of atherosclerosis: a spatial approach to endothelial structure, gene expression, and function. Annals of the New York Academy of Sciences 947, 7-16; discussion 16-17.
De Keulenaer, G.W., Chappell, D.C., Ishizaka, N., Nerem, R.M., Alexander, R.W., and Griendling, K.K. (1998). Oscillatory and steady laminar shear stress differentially affect human endothelial redox state: role of a superoxide-producing NADH oxidase. Circulation Research 82, 1094-1101.
Dekker, R.J., van Thienen, J.V., Rohlena, J., de Jager, S.C., Elderkamp, Y.W., Seppen, J., de Vries, C.J., Biessen, E.A., van Berkel, T.J., Pannekoek, H., et al. (2005). Endothelial KLF2 links local arterial shear stress levels to the expression of vascular tone-regulating genes. American Journal of Pathology 167, 609-618.
Diamond, S.L., Eskin, S.G., and McIntire, L.V. (1989). Fluid flow stimulates tissue plasminogen activator secretion by cultured human endothelial cells. Science (New York, NY 243), 1483-1485.


Dittman, W.A., Kumada, T., and Majerus, P.W. (1989). Transcription of thrombomodulin mRNA in mouse hemangioma cells is increased by cycloheximide and thrombin. Proceedings of the National Academy of Sciences of the United States of America 86, 7179-7182.
Dittman, W.A., Kumada, T., Sadler, J.E., and Majerus, P.W. (1988). The structure and function of mouse thrombomodulin. Phorbol myristate acetate stimulates degradation and synthesis of thrombomodulin without affecting mRNA levels in hemangioma cells. Journal of Biological Chemistry 263, 15815-15822.
Drake, P.M., Gunn, M.D., Charo, I.F., Tsou, C.L., Zhou, Y., Huang, L., and Fisher, S.J. (2001). Human placental cytotrophoblasts attract monocytes and CD56(bright) natural killer cells via the actions of monocyte inflammatory protein 1alpha. Journal of Experimental Medicine 193, 1199-1212.
Dudek, A.Z., Pennell, C.A., Decker, T.D., Young, T.A., Key, N.S., and Slungaard, A. (1997). Platelet factor 4 binds to glycanated forms of thrombomodulin and to protein C. A potential mechanism for enhancing generation of activated protein C. Journal of Biological Chemistry 272, 31785-31792.
Esmon, C. (2005). Do-all receptor takes on coagulation, inflammation. Nature Medicine 11, 475-477.
Esmon, C.T. (2006). Inflammation and the activated protein C anticoagulant pathway. Seminars in Thrombosis and Hemostasis 32 Suppl 1, 49-60.
Esmon, C.T., Fukudome, K., Mather, T., Bode, W., Regan, L.M., Stearns-Kurosawa, D.J., and Kurosawa, S. (1999a). Inflammation, sepsis, and coagulation. Haematologica 84, 254-259.
Esmon, C.T., Gu, J.M., Xu, J., Qu, D., Stearns-Kurosawa, D.J., and Kurosawa, S. (1999b). Regulation and functions of the protein C anticoagulant pathway. Haematologica 84, 363-368.
Fairlie, W.D., Moore, A.G., Bauskin, A.R., Russell, P.K., Zhang, H.P., and Breit, S.N. (1999). MIC-1 is a novel TGF-beta superfamily cytokine associated with macrophage activation. Journal of Leukocyte Biology 65, 2-5.
Faxon, D.P., Creager, M.A., Smith, S.C., Jr., Pasternak, R.C., Olin, J.W., Bettmann, M.A., Criqui, M.H., Milani, R.V., Loscalzo, J., Kaufman, J.A., et al. (2004). Atherosclerotic Vascular Disease Conference: Executive summary: Atherosclerotic Vascular Disease Conference proceeding for healthcare professionals from a special writing group of the American Heart Association. Circulation 109, 2595-2604.
Frangos, J.A., Eskin, S.G., McIntire, L.V., and Ives, C.L. (1985). Flow effects on prostacyclin production by cultured human endothelial cells. Science (New York, NY 227, 1477-1479.
Furchgott, R.F., and Zawadzki, J.V. (1980). The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376.
Gabriels, J.E., and Paul, D.L. (1998). Connexin43 is highly localized to sites of disturbed flow in rat aortic endothelium but connexin37 and connexin40 are more uniformly distributed. Circulation Research 83, 636-643.
Gerthoffer, W.T. (2007). Mechanisms of vascular smooth muscle cell migration. Circulation Research 100, 607-621.
Gimbrone, M.A., Jr., Topper, J.N., Nagel, T., Anderson, K.R., and Garcia-Cardena, G. (2000). Endothelial dysfunction, hemodynamic forces, and atherogenesis. Annals of the New York Academy of Sciences 902, 230-239; discussion 239-240.
Gow, A.J. (2005). Nitric oxide, hemoglobin, and hypoxic vasodilation. American Journal of Respiratory Cell and Molecular Biology 32, 479-482.
Greenwood, J.A., Theibert, A.B., Prestwich, G.D., and Murphy-Ullrich, J.E. (2000). Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin. Journal of Cell Biology 150, 627-642.
Grey, S.T., Csizmadia, V., and Hancock, W.W. (1998). Differential effect of tumor necrosis factor-alpha on thrombomodulin gene expression by human monocytoid (THP-1) cell versus endothelial cells. International Journal of Hematology 67, 53-62.
Hamada, H., Ishii, H., Sakyo, K., Horie, S., Nishiki, K., and Kazama, M. (1995). The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood 86, 225-233.
Han, D.C., Shen, T.L., and Guan, J.L. (2000). Role of Grb7 targeting to focal contacts and its phosphorylation by focal adhesion kinase in regulation of cell migration. Journal of Biological Chemistry 275, 28911-28917.
Hansson, G.K. (2005). Inflammation, atherosclerosis, and coronary artery disease. New England Journal of Medicine 352, 1685-1695.
Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B., and Drexler, H. (2003). Role of oxidative stress in atherosclerosis. American Journal of Cardiology 91, 7A-11A.
Hennig, B., and Boissonneault, G.A. (1987). Cholestan-3 beta,5 alpha,6 beta-triol decreases barrier function of cultured endothelial cell monolayers. Atherosclerosis 68, 255-261.
Hess, D.T., Matsumoto, A., Kim, S.O., Marshall, H.E., and Stamler, J.S. (2005). Protein S-nitrosylation: purview and parameters. Nature Reviews 6, 150-166.
Hirokawa, K., and Aoki, N. (1991). Regulatory mechanisms for thrombomodulin expression in human umbilical vein endothelial cells in vitro. Journal of Cellular Physiology 147, 157-165.
Hobson, B., and Denekamp, J. (1984). Endothelial proliferation in tumours and normal tissues: continuous labelling studies. British Journal of Cancer 49, 405-413.
Horie, S., Ishii, H., Matsumoto, F., Kusano, M., Kizaki, K., Matsuda, J., and Kazama, M. (2001). Acceleration of thrombomodulin gene transcription by retinoic acid: retinoic acid receptors and Sp1 regulate the promoter activity through interactions with two different sequences in the 5''-flanking region of human gene. Journal of Biological Chemistry 276, 2440-2450.
Huddleson, J.P., Ahmad, N., and Lingrel, J.B. (2006). Up-regulation of the KLF2 transcription factor by fluid shear stress requires nucleolin. Journal of Biological Chemistry 281, 15121-15128.
Huddleson, J.P., Ahmad, N., Srinivasan, S., and Lingrel, J.B. (2005). Induction of KLF2 by fluid shear stress requires a novel promoter element activated by a phosphatidylinositol 3-kinase-dependent chromatin-remodeling pathway. Journal of Biological Chemistry 280, 23371-23379.
Iino, S., Abeyama, K., Kawahara, K., Yamakuchi, M., Hashiguchi, T., Matsukita, S., Yonezawa, S., Taniguchi, S., Nakata, M., Takao, S., et al. (2004). The antimetastatic role of thrombomodulin expression in islet cell-derived tumors and its diagnostic value. Clin Cancer Res 10, 6179-6188.
Imada, S., Yamaguchi, H., Nagumo, M., Katayanagi, S., Iwasaki, H., and Imada, M. (1990). Identification of fetomodulin, a surface marker protein of fetal development, as thrombomodulin by gene cloning and functional assays. Developmental Biology 140, 113-122.
Ingber, D. (1998). In search of cellular control: signal transduction in context. Journal of Cellular Biochemistry 30-31, 232-237.
Ishida, T., Takahashi, M., Corson, M.A., and Berk, B.C. (1997). Fluid shear stress-mediated signal transduction: how do endothelial cells transduce mechanical force into biological responses? Annals of the New York Academy of Sciences 811, 12-23; discussion 23-14.
Ishii, H., Nakano, M., Tsubouchi, J., Ishikawa, T., Uchiyama, H., Hiraishi, S., Tahara, C., Miyajima, Y., and Kazama, M. (1990). Establishment of enzyme immunoassay of human thrombomodulin in plasma and urine using monoclonal antibodies. Thrombosis and Haemostasis 63, 157-162.
Ishii, H., Tezuka, T., Ishikawa, H., Takada, K., Oida, K., and Horie, S. (2003). Oxidized phospholipids in oxidized low-density lipoprotein down-regulate thrombomodulin transcription in vascular endothelial cells through a decrease in the binding of RARbeta-RXRalpha heterodimers and Sp1 and Sp3 to their binding sequences in the TM promoter. Blood 101, 4765-4774.
Kaczynski, J., Cook, T., and Urrutia, R. (2003). Sp1- and Kruppel-like transcription factors. Genome Biology 4, 206.
Kay, B.K., Williamson, M.P., and Sudol, M. (2000). The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains. FASEB Journal 14, 231-241.
Kinlay, S., Libby, P., and Ganz, P. (2001). Endothelial function and coronary artery disease. Current Opinion in Lipidology 12, 383-389.
Kosonen, O., Kankaanranta, H., Uotila, J., and Moilanen, E. (2000). Inhibition by nitric oxide-releasing compounds of E-selectin expression in and neutrophil adhesion to human endothelial cells. European Journal of Pharmacology 394, 149-156.
Koyama, T., Parkinson, J.F., Aoki, N., Bang, N.U., Muller-Berghaus, G., and Preissner, K.T. (1991). Relationship between post-translational glycosylation and anticoagulant function of secretable recombinant mutants of human thrombomodulin. British Journal of Haematology 78, 515-522.
Kuchan, M.J., Jo, H., and Frangos, J.A. (1994). Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells. The American Journal of Physiology 267, C753-758.
Kuo, C.T., Veselits, M.L., Barton, K.P., Lu, M.M., Clendenin, C., and Leiden, J.M. (1997). The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes & Development 11, 2996-3006.
Kwon, M.S., Hori, T., Okada, M., and Fukagawa, T. (2007). CENP-C Is Involved in Chromosome Segregation, Mitotic Checkpoint Function, and Kinetochore Assembly. Molecular Biology of The Cell 18, 2155-2168.
Lager, D.J., Callaghan, E.J., Worth, S.F., Raife, T.J., and Lentz, S.R. (1995). Cellular localization of thrombomodulin in human epithelium and squamous malignancies. American Journal of Pathology 146, 933-943.
Lahera, V., Goicoechea, M., de Vinuesa, S.G., Miana, M., de las Heras, N., Cachofeiro, V., and Luno, J. (2007). Endothelial dysfunction, oxidative stress and inflammation in atherosclerosis: beneficial effects of statins. Current Medicinal Chemistry 14, 243-248.
Langille, B.L. (1996). Arterial remodeling: relation to hemodynamics. Canadian Journal of Physiology and Pharmacology 74, 834-841.
Langille, B.L., and O''Donnell, F. (1986). Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science (New York, NY 231, 405-407.
Lee, J.O., Yang, H., Georgescu, M.M., Di Cristofano, A., Maehama, T., Shi, Y., Dixon, J.E., Pandolfi, P., and Pavletich, N.P. (1999). Crystal structure of the PTEN tumor suppressor: implications for its phosphoinositide phosphatase activity and membrane association. Cell 99, 323-334.
Li, D.M., and Sun, H. (1997). TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Research 57, 2124-2129.
Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., et al. (1997). PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (New York, NY 275, 1943-1947.
Li, Y.H., Shi, G.Y., and Wu, H.L. (2006). The role of thrombomodulin in atherosclerosis: from bench to bedside. Cardiovascular & Hematological Agents in Medicinal Chemistry 4, 183-187.
Libby, P. (2001). Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 104, 365-372.
Libby, P. (2002). Inflammation in atherosclerosis. Nature 420, 868-874.
Libby, P., Ridker, P.M., and Maseri, A. (2002). Inflammation and atherosclerosis. Circulation 105, 1135-1143.
Liliental, J., Moon, S.Y., Lesche, R., Mamillapalli, R., Li, D., Zheng, Y., Sun, H., and Wu, H. (2000). Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases. Current Biology 10, 401-404.
Lin, Z., Hamik, A., Jain, R., Kumar, A., and Jain, M.K. (2006). Kruppel-like factor 2 inhibits protease activated receptor-1 expression and thrombin-mediated endothelial activation. Arteriosclerosis, Thrombosis, and Vascular Biology 26, 1185-1189.
Lin, Z., Kumar, A., SenBanerjee, S., Staniszewski, K., Parmar, K., Vaughan, D.E., Gimbrone, M.A., Jr., Balasubramanian, V., Garcia-Cardena, G., and Jain, M.K. (2005). Kruppel-like factor 2 (KLF2) regulates endothelial thrombotic function. Circulation Research 96, e48-57.
Lloyd-Jones, D.M., Larson, M.G., Beiser, A., and Levy, D. (1999). Lifetime risk of developing coronary heart disease. Lancet 353, 89-92.
Lusis, A.J. (2000). Atherosclerosis. Nature 407, 233-241.
Malek, A.M., Alper, S.L., and Izumo, S. (1999). Hemodynamic shear stress and its role in atherosclerosis. Jama 282, 2035-2042.
Malek, A.M., Jackman, R., Rosenberg, R.D., and Izumo, S. (1994). Endothelial expression of thrombomodulin is reversibly regulated by fluid shear stress. Circulation Research 74, 852-860.
Marathe, S., Kuriakose, G., Williams, K.J., and Tabas, I. (1999). Sphingomyelinase, an enzyme implicated in atherogenesis, is present in atherosclerotic lesions and binds to specific components of the subendothelial extracellular matrix. Arteriosclerosis, Thrombosis, and Vascular Biology 19, 2648-2658.
Marshall, H.E., Merchant, K., and Stamler, J.S. (2000). Nitrosation and oxidation in the regulation of gene expression. FASEB Journal 14, 1889-1900.
Maruyama, I., Bell, C.E., and Majerus, P.W. (1985). Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. Journal of Cell Biology 101, 363-371.
Mazzone, T., Jensen, M., and Chait, A. (1983). Human arterial wall cells secrete factors that are chemotactic for monocytes. Proceedings of the National Academy of Sciences of the United States of America 80, 5094-5097.
McCaffrey, T.A., Fu, C., Du, B., Eksinar, S., Kent, K.C., Bush, H., Jr., Kreiger, K., Rosengart, T., Cybulsky, M.I., Silverman, E.S., et al. (2000). High-level expression of Egr-1 and Egr-1-inducible genes in mouse and human atherosclerosis. Journal of Clinical Investigation 105, 653-662.
Michel, T., and Feron, O. (1997). Nitric oxide synthases: which, where, how, and why? Journal of Clinical Investigation 100, 2146-2152.
Mocellin, S., Bronte, V., and Nitti, D. (2007). Nitric oxide, a double edged sword in cancer biology: searching for therapeutic opportunities. Medicinal Research Reviews 27, 317-352.
Moore, K.L., Esmon, C.T., and Esmon, N.L. (1989). Tumor necrosis factor leads to the internalization and degradation of thrombomodulin from the surface of bovine aortic endothelial cells in culture. Blood 73, 159-165.


Morawietz, H., Talanow, R., Szibor, M., Rueckschloss, U., Schubert, A., Bartling, B., Darmer, D., and Holtz, J. (2000). Regulation of the endothelin system by shear stress in human endothelial cells. Journal of Physiology 525 Pt 3, 761-770.
Myers, M.P., Pass, I., Batty, I.H., Van der Kaay, J., Stolarov, J.P., Hemmings, B.A., Wigler, M.H., Downes, C.P., and Tonks, N.K. (1998). The lipid phosphatase activity of PTEN is critical for its tumor supressor function. Proceedings of the National Academy of Sciences of the United States of America 95, 13513-13518.
Myers, M.P., Stolarov, J.P., Eng, C., Li, J., Wang, S.I., Wigler, M.H., Parsons, R., and Tonks, N.K. (1997). P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proceedings of the National Academy of Sciences of the United States of America 94, 9052-9057.
Noble, M., and Dietrich, J. (2004). The complex identity of brain tumors: emerging concerns regarding origin, diversity and plasticity. Trends in Neurosciences 27, 148-154.
Ohji, T., Urano, H., Shirahata, A., Yamagishi, M., Higashi, K., Gotoh, S., and Karasaki, Y. (1995). Transforming growth factor beta 1 and beta 2 induce down-modulation of thrombomodulin in human umbilical vein endothelial cells. Thrombosis and Haemostasis 73, 812-818.
Ohlin, A.K., Holm, J., and Hillarp, A. (2004). Genetic variation in the human thrombomodulin promoter locus and prognosis after acute coronary syndrome. Thrombosis Research 113, 319-326.
Okutani, D., Lodyga, M., Han, B., and Liu, M. (2006). Src protein tyrosine kinase family and acute inflammatory responses. Am J Physiol Lung Cell Mol Physiol 291, L129-141.
Ortiz, P.A., and Garvin, J.L. (2003). Trafficking and activation of eNOS in epithelial cells. Acta Physiologica Scandinavica 179, 107-114.
Paramio, J.M., Navarro, M., Segrelles, C., Gomez-Casero, E., and Jorcano, J.L. (1999). PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 18, 7462-7468.
Park, H.J., Zhang, Y., Georgescu, S.P., Johnson, K.L., Kong, D., and Galper, J.B. (2006). Human umbilical vein endothelial cells and human dermal microvascular endothelial cells offer new insights into the relationship between lipid metabolism and angiogenesis. Stem Cell Reviews 2, 93-102.


Parmar, K.M., Larman, H.B., Dai, G., Zhang, Y., Wang, E.T., Moorthy, S.N., Kratz, J.R., Lin, Z., Jain, M.K., Gimbrone, M.A., Jr., et al. (2006). Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2. The Journal of Clinical Investigation 116, 49-58.
Qiao, J.H., Tripathi, J., Mishra, N.K., Cai, Y., Tripathi, S., Wang, X.P., Imes, S., Fishbein, M.C., Clinton, S.K., Libby, P., et al. (1997). Role of macrophage colony-stimulating factor in atherosclerosis: studies of osteopetrotic mice. The American Journal of Pathology 150, 1687-1699.
Raife, T.J., Lager, D.J., Madison, K.C., Piette, W.W., Howard, E.J., Sturm, M.T., Chen, Y., and Lentz, S.R. (1994). Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. The Journal of Clinical Investigation 93, 1846-1851.
Resnick, N., Yahav, H., Shay-Salit, A., Shushy, M., Schubert, S., Zilberman, L.C., and Wofovitz, E. (2003). Fluid shear stress and the vascular endothelium: for better and for worse. Progress in Biophysics and Molecular Biology 81, 177-199.
Rosenfeld, C.S. (1992). Effects of L-leucyl-L-leucine methyl ester on human marrow and protection of progenitor cells by IL-1. International Journal of Cell Cloning 10, 249-253.
Ross, R. (1999). Atherosclerosis--an inflammatory disease. The New England Journal of Medicine 340, 115-126.
Sandusky, G., Berg, D.T., Richardson, M.A., Myers, L., and Grinnell, B.W. (2002). Modulation of thrombomodulin-dependent activation of human protein C through differential expression of endothelial Smads. Journal of Biological Chemistry 277, 49815-49819.
Sato, Y., Hamanaka, R., Ono, J., Kuwano, M., Rifkin, D.B., and Takaki, R. (1991). The stimulatory effect of PDGF on vascular smooth muscle cell migration is mediated by the induction of endogenous basic FGF. Biochemical and Biophysical Research Communications 174, 1260-1266.
Sen-Banerjee, S., Mir, S., Lin, Z., Hamik, A., Atkins, G.B., Das, H., Banerjee, P., Kumar, A., and Jain, M.K. (2005). Kruppel-like factor 2 as a novel mediator of statin effects in endothelial cells. Circulation 112, 720-726.
Shen, W.H., Balajee, A.S., Wang, J., Wu, H., Eng, C., Pandolfi, P.P., and Yin, Y. (2007). Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell 128, 157-170.
Smithies, O., and Maeda, N. (1995). Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension. Proceedings of the National Academy of Sciences of the United States of America 92, 5266-5272.
Sokabe, T., Yamamoto, K., Ohura, N., Nakatsuka, H., Qin, K., Obi, S., Kamiya, A., and Ando, J. (2004). Differential regulation of urokinase-type plasminogen activator expression by fluid shear stress in human coronary artery endothelial cells. American Journal of Physiology 287, H2027-2034.
Steck, P.A., Pershouse, M.A., Jasser, S.A., Yung, W.K., Lin, H., Ligon, A.H., Langford, L.A., Baumgard, M.L., Hattier, T., Davis, T., et al. (1997). Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nature Genetics 15, 356-362.
Stephens, N.G., Parsons, A., Schofield, P.M., Kelly, F., Cheeseman, K., and Mitchinson, M.J. (1996). Randomised controlled trial of vitamin E in patients with coronary disease: Cambridge Heart Antioxidant Study (CHAOS). Lancet 347, 781-786.
Stone, P.H., Coskun, A.U., Yeghiazarians, Y., Kinlay, S., Popma, J.J., Kuntz, R.E., and Feldman, C.L. (2003). Prediction of sites of coronary atherosclerosis progression: In vivo profiling of endothelial shear stress, lumen, and outer vessel wall characteristics to predict vascular behavior. Current Opinion in Cardiology 18, 458-470.
Suzuki, K., Kusumoto, H., Deyashiki, Y., Nishioka, J., Maruyama, I., Zushi, M., Kawahara, S., Honda, G., Yamamoto, S., and Horiguchi, S. (1987). Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO Journal 6, 1891-1897.
Suzuki, T., Aizawa, K., Matsumura, T., and Nagai, R. (2005). Vascular implications of the Kruppel-like family of transcription factors. Arteriosclerosis, Thrombosis, and Vascular Biology 25, 1135-1141.
Teasdale, M.S., Bird, C.H., and Bird, P. (1994). Internalization of the anticoagulant thrombomodulin is constitutive and does not require a signal in the cytoplasmic domain. Immunology and Cell Biology 72, 480-488.
Tohda, G., Oida, K., Okada, Y., Kosaka, S., Okada, E., Takahashi, S., Ishii, H., and Miyamori, I. (1998). Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular smooth muscle cells. Arteriosclerosis, Thrombosis, and Vascular Biology 18, 1861-1869.
Torres, J., and Pulido, R. (2001). The tumor suppressor PTEN is phosphorylated by the protein kinase CK2 at its C terminus. Implications for PTEN stability to proteasome-mediated degradation. Journal of Biological Chemistry 276, 993-998.

Trotman, L.C., Wang, X., Alimonti, A., Chen, Z., Teruya-Feldstein, J., Yang, H., Pavletich, N.P., Carver, B.S., Cordon-Cardo, C., Erdjument-Bromage, H., et al. (2007). Ubiquitination regulates PTEN nuclear import and tumor suppression. Cell 128, 141-156.
Tzima, E., Irani-Tehrani, M., Kiosses, W.B., Dejana, E., Schultz, D.A., Engelhardt, B., Cao, G., DeLisser, H., and Schwartz, M.A. (2005). A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437, 426-431.
Van de Wouwer, M., and Conway, E.M. (2004). Novel functions of thrombomodulin in inflammation. Critical Care Medicine 32, S254-261.
Van de Wouwer, M.a.C., EM. (2003). thrombomodulin: a regulator of coagulation, fibrinolysis, inflammation and cell proliferation. Thrombosis-Fundamental and Clinical Aspects. (Leuven: Leuven University Press).
van Thienen, J.V., Fledderus, J.O., Dekker, R.J., Rohlena, J., van Ijzendoorn, G.A., Kootstra, N.A., Pannekoek, H., and Horrevoets, A.J. (2006). Shear stress sustains atheroprotective endothelial KLF2 expression more potently than statins through mRNA stabilization. Cardiovascular Research 72, 231-240.
Waite, K.A., and Eng, C. (2002). Protean PTEN: form and function. American Journal of Human Genetics 70, 829-844.
Wang, N., Miao, H., Li, Y.S., Zhang, P., Haga, J.H., Hu, Y., Young, A., Yuan, S., Nguyen, P., Wu, C.C., et al. (2006). Shear stress regulation of Kruppel-like factor 2 expression is flow pattern-specific. Biochemical and Biophysical Research Communications 341, 1244-1251.
Wani, M.A., Wert, S.E., and Lingrel, J.B. (1999). Lung Kruppel-like factor, a zinc finger transcription factor, is essential for normal lung development. The Journal of Biological Chemistry 274, 21180-21185.
Weiler, H. (2004). Mouse models of thrombosis: thrombomodulin. Thrombosis and Haemostasis 92, 467-477.
Weiler, H., Lindner, V., Kerlin, B., Isermann, B.H., Hendrickson, S.B., Cooley, B.C., Meh, D.A., Mosesson, M.W., Shworak, N.W., Post, M.J., et al. (2001). Characterization of a mouse model for thrombomodulin deficiency. Arteriosclerosis, Thrombosis, and Vascular Biology 21, 1531-1537.
Weisel, J.W., Nagaswami, C., Young, T.A., and Light, D.R. (1996). The shape of thrombomodulin and interactions with thrombin as determined by electron microscopy. Journal of Biological Chemistry 271, 31485-31490.

Weng, L., Brown, J., and Eng, C. (2001). PTEN induces apoptosis and cell cycle arrest through phosphoinositol-3-kinase/Akt-dependent and -independent pathways. Human Molecular Genetics 10, 237-242.
Werdich, X.Q., and Penn, J.S. (2005). Src, Fyn and Yes play differential roles in VEGF-mediated endothelial cell events. Angiogenesis 8, 315-326.
Wu, J., and Lingrel, J.B. (2004). KLF2 inhibits Jurkat T leukemia cell growth via upregulation of cyclin-dependent kinase inhibitor p21WAF1/CIP1. Oncogene 23, 8088-8096.
Wu, R., Huang, Y.H., Elinder, L.S., and Frostegard, J. (1998). Lysophosphatidylcholine is involved in the antigenicity of oxidized LDL. Arteriosclerosis, Thrombosis, and Vascular Biology 18, 626-630.
Wu, X., Hepner, K., Castelino-Prabhu, S., Do, D., Kaye, M.B., Yuan, X.J., Wood, J., Ross, C., Sawyers, C.L., and Whang, Y.E. (2000). Evidence for regulation of the PTEN tumor suppressor by a membrane-localized multi-PDZ domain containing scaffold protein MAGI-2. Proceedings of the National Academy of Sciences of the United States of America 97, 4233-4238.
Yang, L., Manithody, C., Walston, T.D., Cooper, S.T., and Rezaie, A.R. (2003). Thrombomodulin enhances the reactivity of thrombin with protein C inhibitor by providing both a binding site for the serpin and allosterically modulating the activity of thrombin. Journal of Biological Chemistry 278, 37465-37470.
王安姬 (2006). 內皮細胞中凝血酶調節素TM及轉錄因子KLF2受剪力調控及其訊息傳遞路徑之探討 (國立台灣大學化學工程研究所碩士學位論文).
蕭錫閩 (2005). 凝血酶調節素各功能區域對於抗發炎反應之探討 (國立成功大學生物化學暨分子生物研究所碩士論文).