Homoeostatic regulation of cytokines to retard liver fibrosis 1

HOMEOSTATIC REGULATION OF CYTOKINES TO RETARD LIVER FIBROSIS BALAKRISHNAN CHAKRPANI NARMADA NATIONAL UNIVERSITY OF SINGAPORE 2012 HOMEOSTATIC REGULATION OF CYTOKINES TO RETARD LIVER FIBROSIS BALAKRISHNAN CHAKRAPANI NARMADA (B. Tech. (Biotechnology), Vellore Institute of Technology, Vellore, India) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS Graduate School for Integrative Sciences and Engineering NATIONAL UNIVERSITY OF SINGAPORE 2012 ! Declaration! ! ! ! I!hereby!declare!that!the!thesis!is!my!original! ! work!and!it!has!been!written!by!me!in!its! ! entirety.!I!have!duly!acknowledged!all!the! ! sources!of!information,!which!have!been!used!in! ! the!thesis.! ! ! ! ! This!thesis!has!also!not!been!submitted!for!any! ! degree!in!any!university!previously.! ! ! ! ! _________________! ! Balakrishnan!Chakrapani!Narmada! ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my father, Chakrapani, for his neverending faith, constant encouragement, support, and guidance and for being a wonderful dad to me. I thank my mother, Uma for constantly nagging and taking care of my health and my food habits, for her encouragement, her support in all of my decisions, her patience when I extended my course and for just being her. I am very lucky to have such wonderful parents and their immense love, and I sincerely hope that I can live up to their dreams. Special thanks to my fiancé, Koushik for his love, indefinite support, sanity, and understanding throughout the years and for always being there for me. I thank my Sister, Sinduja for always being there for our family and for me whenever I want a reprieve from the daily rush. I would like to thank my grandparents, Balakrishnan and Gnanambal and my brother, Hari for their constant support and love. I would like to thank my uncle, Babu and aunt, Indu for their immense support, encouragement and guidance and their beautiful kids for their love. I would like to thank my uncle Jayaraj for his constant encouragement. I thank my supervisor Dr. Hanry Yu for his guidance, immense support, scientific discussions and teachings on matrix management, team building, and project management and for providing me this wonderful opportunity of working in a multidisciplinary laboratory. I thank Dr. Lisa Tucker-Kellogg for her patience, support, guidance, scientific discussions and teaching the art of scientific writing. I would like to thank my friends, Abhishek and Lakshmi for their scientific discussions, immense help in animal handling and their patience in hearing me out during the difficult times. I would also like to thank them, Justin, Inn Chuan, Yee Han, George i Anene, Madumathi, Siow Thing, Derek, Ali and Yi-Chin for their constant support and the fun-filled times. I sincerely thank our lab executive, Phoebe for her immense support and patience. I would like to express my sincere gratitude towards NGS for this opportunity and for their funding support throughout my doctoral research. I would also like to thank NGS admin officers, Irene and Jenny for their immense help in various admin-related matters. Finally, I would like to thank God for providing me the strength and courage to pursue my dreams. ii TABLE OF CONTENTS TABLE OF CONTENTS……………………………………………………… iii SUMMARY…………………………………………………………………… viii LIST OF ABBREVIATIONS………………………………………………… x LIST OF TABLES………………………………………………………………xii LIST OF FIGURES…………………………………………………………….xiii 1. INTRODUCTION 1.1. LIVER STRUCTURE AND FUNCTION……………………………….1 1.1.1. Anatomy Of The Liver………………………………………………1 1.1.2. Liver Functions And The Importance of Zonation………………… 1.1.3. Cells In The Sinusoidal Lumen…………………………………… .5 1.1.3.1.Hepatocytes………………………………………………………5 1.1.3.1.1. Hepatocyte functions…………………………………… 1.1.3.2.Hepatic Stellate cells…………………………………………… .7 1.1.3.3.Kupffer Cells…………………………………………………… .9 1.1.3.4.Sinusoidal Endothelial Cells………………………………………9 1.1.3.4.1. The functional role of liver sinusoidal endothelia……….12 1.2. FIBROSIS………………………………………………………………….13 1.2.1. Etiology…………………………………………………………… .15 1.2.2. Homeostatic Regulation Of Cytokines During Liver Repair/Wound Healing……………………………………………….16 1.2.2.1. Hepatic Fibrosis And Myofibroblasts………………………… .17 1.2.2.2. Activation of HSCs into myofibroblasts……………………… .17 1.2.2.2.1. Role of retinoids in the proliferation of activated HSCs 19 iii 1.2.2.2.2. Soluble factors mediating the activation of HSCs………19 1.2.2.3. Importance Of TGF-β1 Activation Pathway In HSC Activation.20 1.2.2.4. Microvascular changes………………………………………….21 1.3. HEPATOCYTE GROWTH FACTOR, HEPATIC REGENERATION & LIVER FIBROSIS 1.3.1. Hepatocyte Transplantation As A Therapy For Liver Fibrosis…… .23 1.3.2. Hepatocyte Growth Factor & Structure…………………………… .24 1.3.3. Current mechanistic understanding of the anti-fibrotic role of HGF .25 1.4. THERAPEUTIC STRATEGIES FOR THE TREATMENT OF LIVER FIBROSIS 1.4.1. Treatments Currently Available In The Clinics & In Clinical Trials 26 1.4.2. Therapies Targeting HSCs And HSC activation…………………….28 1.4.3. Blood Barriers Against Hepatic Gene Therapy…………………… .29 1.4.4. Anti-Fibrotic Therapies Specifically Targeting The Stellate Cells….30 2. NOVEL REGULATORY ROLE OF HGF ON TGF-β1 ACTIVATION DURING LIVER FIBROSIS 2.1. AIMS & OBJECTIVES………………………………………………… .34 2.2. MATERIALS & METHODS 2.2.1. Cell Culture Models………………………………………………….37 2.2.2. Inhibitors…………………………………………………………… 39 2.2.3. Picogreen assay to measure hepatocyte proliferation……………… 39 2.2.4. ELISA……………………………………………………………… 39 2.2.5. Western Blot…………………………………………………………40 2.2.6. Gene Expression – RT-PCR…………………………………………41 iv 2.2.7. Statistical Analysis………………………………………………… .42 2.3. RESULTS 2.3.1. HGF increased total plasmin levels through hepatocyte proliferation and decreased expression of pro-fibrotic genes……………………… .42 2.3.2. Plasmin mediated the HGF-induced decrease of active TGF-β1 and Collagen I levels……………………………………………………… 45 2.3.3. HGF antagonized TSP-1-dependent TGF-β1 activation…………….50 2.4. DISCUSSION………………………………………………………………55 3. HSC-TARGETED DELIVERY OF HGF TRANSGENE ADMINISTERED VIA BILE DUCT INFUSION ENHANCES ITS LOCALIZATION AT FIBROTIC FOCI & AMELIORATES DMN-INDUCED LIVER FIBROSIS 3.1. AIMS & OBJECTIVES….…………………………………………… …60 3.2. MATERIALS & METHODS 3.2.1. In Vitro Cultures…………………………………………………… 62 3.2.2. Meaurement Of Hepatocyte Proliferation………………………… .63 3.2.3. Transgene Validation By Transfection In HEK-293T Cells……… .63 3.2.4. Transgene Construction And Encapsulation In Vitamin A-coupled Liposomes…………………………………………………………… .63 3.2.5. Protein Measurements By Western Blot…………………………….65 3.2.6. DMN-Induced Liver Disease……………………………………… 66 3.2.7. Retrograde Intrabiliary Infusion…………………………………… 66 3.2.8. Gene Expression Analysis – RT-PCR………………………………67 3.2.9. Active TGF-β1 Measurement……………………………………….67 3.2.10. Liver Protein Levels…………………………………………………67 v 3.2.11. Collagen Imaging – Non Linear Microscopy & Image Acquisition .68 3.2.12. Immunohistochemistry………………………………………………68 3.2.13. Immunofluorescence……………………………………………… .68 3.2.14. Scanning Electron Microscopy for SECs……………………………69 3.2.15. Statistical Analysis………………………………………………… 69 3.3. RESULTS 3.3.1. pDsRed2-HGF Gene Construction & In Vitro Validation………… .69 3.3.2. Effects of Vitamin A-liposome-HGF on fibrotic cultures in vitro 71 3.3.3. Establishment Of DMN-induced liver fibrosis…………… .……….73 3.3.4. Vascular Dysfunction In DMN-Induced Fibrotic Livers…………….77 3.3.5. Regression of DMN-Induced Liver Fibrosis After VALH Treatment79 3.3.6. Localized Delivery of HGF Transgene To Areas Expressing α-SMA79 3.3.7. VALH Treatment Caused A Decline In Serum Markers Of Fibrosis 82 3.3.8. VALH Treatment Improved Structural Markers Of Fibrosis……… 83 3.3.9. Enhanced Spatial Localization Of HGF Gene Within The Fibrotic Foci Causes Decline In HSC-Specific Markers Implicated In Fibrogenesis .85 3.4. DISCUSSION………………………………………………………………88 4. CONCLUSION…………………………………………………………………92 5. RECOMMENDATIONS FOR FUTURE RESEARCH……………………95 5.1. ROLE OF TGF-β CO-REPRESSOR SNON IN THE HGF- DEPENDENT INHIBITION OF TGF-β1 ACTIVATION…………… 95 vi 5.2. REGRESSION OF LIVER FIBROSIS IN TAA-INDUCED FIBROTIC RATS AFTER RETROGRADE INTRABILIARY INFUSION OF VALH PARTICLES…………………………………………………… 97 5.3. ALTERNATIVE SOURCE TRANSPLANTATION OF PURPOSES HEPATOCYTES IN END-STAGE FOR LIVER DISEASES……………………………………………………………… .100 5.3.1. iPSC-Derived Hepatocytes……………………………………… 105 BIBLIOGRAPHY…………………………………………………………………107 APPENDICES……………………………………………………………………… a Publications……………………………………………………………… b Conference Proceedings…………………………………………………… c vii Figure 44: Undifferentiated feeder-free iPSF4 colonies. iPSF4 cell colonies maintained in mTESR1 medium showing undifferentiated morphology under phase contrast microscope (10x magnification). We cultured the iPS cells in matrigel-coated dishes in APEL medium and treated them with different cytokines for different time periods as described in Fig. 45. Oncostatin-M Figure 45: Schematic representation of the protocol for directed differentiation of pluripotent stem cells into mature hepatocytes. Protocol adapted from (182). Initial phase of differentiation until day 10 is to induce the pluripotent stem cells into the definitive endoderm lineage by inducing the Activin/Wnt signaling pathway. The   102   canonical Wnt signaling involves Wnt proteins and their interactions with cell-surface receptors of the Frizzled family on target cells and further signaling that regulates the amount of B-catenin that enters the nucleus in turn regulates physiological responses such as cell growth and morphogensis (187). As seen in Fig. 46, 10 days from start of differentiation there was a strong increase in the marker for endoderm, Foxa2 as compared to the ectoderm or mesoderm markers Pax6 and Brachury/T respectively in feeder-free cultures. A   103   Fold change over day control (Normalized to GAPDH) B Day 100 Day 10 80 60 40 20 10 -5 -10 Oct3/4 Foxa2 T Pax6 Figure 46: Definitive endoderm induction of hiPSCs. Morphology of iPSF4 cell colonies in feeder and feeder-free configurations (A; 4x magnification). At day and day 10, the feeder-free cultures show a gradual progression in definite endoderm marker Foxa2 The next phase of hepatic induction leads to highly specific epithelial morphology similar to mature hepatocytes (Fig. 47A) and significant increases in the hepatocytespecific genes such as albumin, AAT, HNF4A and CYP3A4 (Fig. 47B). A   Feeder culture Feeder-free culture 104   B Figure 47: Expression of hepatocyte-like markers in differentiated hiPSCs by day 20. Photomicrographs showing hepatocyte-like morphology of differentiated hiPSCs (A; upper panel: 4x magnification, lower panel: 40x magnification). Increased expression of hepatocyte specific genes as assessed from RT-PCR measurements (B). We also observed a significant increase in the expression of hepatic markers albumin and MRP-2 (Fig. 48) and increase in albumin secretion (140.57 ± 7.33ng/million cells) in the hepatocyte-like cells derived from hiPSCs. Figure 48: Expression of mature hepatic markers by the hepatocyte-like cells derived from hiPSCs. Albumin (green) and MRP-2 (red). Scale bar: 20 µm Further characterization of the hepatocyte functions at day 20 and testing for safety, efficient integration and function in vivo is required before these cells can be further used as an alternative source for hepatocyte transplantation in clinics.   105   BIBLIOGRAPHY   106   1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.   Junqueira, L., and Carneiro, J. (2003) Organs associated with the digestive tract. in Basic histology: text and atlas (Junqueira, L., and Carneiro, J. eds.), 11th Ed., The McGraw Hill Companies. pp 332-343 Racanelli, V., and Rehermann, B. (2006) Hepatology 43, S54-62 Arias, I. M., Che, M., Gatmaitan, Z., Leveille, C., Nishida, T., and St Pierre, M. (1993) Hepatology 17, 318-329 Stamatoglou, S. C., and Hughes, R. C. (1994) FASEB J 8, 420-427 Michalopoulos, G. K. (2007) J Cell Physiol 213, 286-300 Lake, B. G., Price, R. J., Giddings, A. M., and Walters, D. G. (2009) Methods Mol Biol 481, 47-58 Friedman, S. L. (2000) J Biol Chem 275, 2247-2250 Gandhi, C. R. (2011) Stellate Cells. in Molecular Pathology of Liver Diseases (Monga, S. P. S. ed.), Springer. pp 53-79 Gandhi, C. R. (2011) Kupffer Cells. in Molecular Pathology of Liver Diseases (Monga, S. P. S. ed.), Springer. pp 81-95 Shibayama, Y., and Nakata, K. (1985) Hepatology 5, 643-648 Ueno, T., Bioulac-Sage, P., Balabaud, C., and Rosenbaum, J. (2004) Anat Rec A Discov Mol Cell Evol Biol 280, 868-873 Gandhi, C. R., Berkowitz, D. E., and Watkins, W. D. (1994) Anesthesiology 80, 892-905 Gandhi, C. R., Sproat, L. A., and Subbotin, V. M. (1996) Life Sci 58, 55-62 Aird, W. C. (2007) Circ Res 100, 174-190 Aird, W. C. (2007) Circ Res 100, 158-173 Wisse, E. (1972) J Ultrastruct Res 38, 528-562 Stolz, D. B. (2011) Sinusoidal Endothelial Cells. in Molecular Pathology of Liver Diseases (Monga, S. P. S. ed.), Springer. pp 97-107 Smedsrod, B., Le Couteur, D., Ikejima, K., Jaeschke, H., Kawada, N., Naito, M., Knolle, P., Nagy, L., Senoo, H., Vidal-Vanaclocha, F., and Yamaguchi, N. (2009) Liver Int 29, 490-501 Braet, F., and Wisse, E. (2002) Comp Hepatol 1, Wisse, E., Jacobs, F., Topal, B., Frederik, P., and De Geest, B. (2008) Gene Ther 15, 1193-1199 Wisse, E., De Zanger, R. B., Jacobs, R., and McCuskey, R. S. (1983) Scan Electron Microsc, 1441-1452 Fraser, R., Dobbs, B. R., and Rogers, G. W. (1995) Hepatology 21, 863-874 Wisse, E., De Zanger, R. B., Charels, K., Van Der Smissen, P., and McCuskey, R. S. (1985) Hepatology 5, 683-692 Britton, R. S., and Bacon, B. R. (1999) Alcohol Clin Exp Res 23, 922-925 Friedman, S. L. (1999) Alcohol Clin Exp Res 23, 904-910 Reeves, H. L., and Friedman, S. L. (2002) Front Biosci 7, d808-826 Bataller, R., and Brenner, D. A. (2005) J Clin Invest 115, 209-218 Iredale, J. P. (2007) J Clin Invest 117, 539-548 Schnabl, B., Scholten, D., and Brenner, D. A. (2008) Nat Clin Pract Gastroenterol Hepatol 5, 496-497 Friedman, S. L., and Bansal, M. B. (2006) Hepatology 43, S82-88 Everhart, J. E., and Ruhl, C. E. (2009) Gastroenterology 136, 1134-1144 Lim, Y. S., and Kim, W. R. (2008) Clin Liver Dis 12, 733-746, vii Kisseleva, T., and Brenner, D. A. (2007) J Gastroenterol Hepatol 22 Suppl 1, S73-78 107   34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62.   Mallat, A., Hezode, C., and Lotersztajn, S. (2008) J Hepatol 48, 657-665 Knittel, T., Kobold, D., Piscaglia, F., Saile, B., Neubauer, K., Mehde, M., Timpl, R., and Ramadori, G. (1999) Histochemistry and cell biology 112, 387401 Popper, H., and Uenfriend, S. (1970) Am J Med 49, 707-721 Ley, K. (1996) Cardiovasc Res 32, 733-742 Gressner, A. M. (1995) J Hepatol 22, 28-36 De Minicis, S., Seki, E., Uchinami, H., Kluwe, J., Zhang, Y., Brenner, D. A., and Schwabe, R. F. (2007) Gastroenterology 132, 1937-1946 Leo, M. A., and Lieber, C. S. (1982) N Engl J Med 307, 597-601 Pinzani, M., Gentilini, P., and Abboud, H. E. (1992) J Hepatol 14, 211-220 Senoo, H., and Wake, K. (1985) Lab Invest 52, 182-194 Davis, B. H., Rapp, U. R., and Davidson, N. O. (1991) Biochem J 278 ( Pt 1), 43-47 Gressner, A. M., Lahme, B., and Brenzel, A. (1995) Hepatology 22, 15071518 Hellerbrand, C., Stefanovic, B., Giordano, F., Burchardt, E. R., and Brenner, D. A. (1999) J Hepatol 30, 77-87 Neubauer, K., Kruger, M., Quondamatteo, F., Knittel, T., Saile, B., and Ramadori, G. (1999) J Hepatol 31, 692-702 Prosser, C. C., Yen, R. D., and Wu, J. (2006) World J Gastroenterol 12, 509515 Saile, B., Matthes, N., Knittel, T., and Ramadori, G. (1999) Hepatology 30, 196-202 Armendariz-Borunda, J., Katai, H., Jones, C. M., Seyer, J. M., Kang, A. H., and Raghow, R. (1993) Lab Invest 69, 283-294 Jarvelainen, H. A., Fang, C., Ingelman-Sundberg, M., and Lindros, K. O. (1999) Hepatology 29, 1503-1510 Schnabl, B., Kweon, Y. O., Frederick, J. P., Wang, X. F., Rippe, R. A., and Brenner, D. A. (2001) Hepatology 34, 89-100 Bosch, J. (2007) J Clin Gastroenterol 41 Suppl 3, S247-253 Iwakiri, Y., and Groszmann, R. J. (2007) J Hepatol 46, 927-934 DeLeve, L. D., Wang, X., Hu, L., McCuskey, M. K., and McCuskey, R. S. (2004) Am J Physiol Gastrointest Liver Physiol 287, G757-763 Pinzani, M., and Vizzutti, F. (2008) Clin Liver Dis 12, 901-913, x Popper, H. (1977) Am J Pathol 87, 228-264 Fernandez, M., Semela, D., Bruix, J., Colle, I., Pinzani, M., and Bosch, J. (2009) J Hepatol 50, 604-620 Gieling, R. G., Burt, A. D., and Mann, D. A. (2008) Clin Liver Dis 12, 915937, xi Corpechot, C., Barbu, V., Wendum, D., Kinnman, N., Rey, C., Poupon, R., Housset, C., and Rosmorduc, O. (2002) Hepatology 35, 1010-1021 Lee, J. S., Semela, D., Iredale, J., and Shah, V. H. (2007) Hepatology 45, 817825 Novo, E., Cannito, S., Zamara, E., Valfre di Bonzo, L., Caligiuri, A., Cravanzola, C., Compagnone, A., Colombatto, S., Marra, F., Pinzani, M., and Parola, M. (2007) Am J Pathol 170, 1942-1953 Semela, D., Das, A., Langer, D., Kang, N., Leof, E., and Shah, V. (2008) Gastroenterology 135, 671-679 108   63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85.   Racine-Samson, L., Scoazec, J. Y., D'Errico, A., Fiorentino, M., Christa, L., Moreau, A., Roda, C., Grigioni, W. F., and Feldman, G. (1996) Hepatology 24, 104-113 Hernandez-Gea, V., and Friedman, S. L. (2011) Annu Rev Pathol 6, 425-456 Yasuda, H., Imai, E., Shiota, A., Fujise, N., Morinaga, T., and Higashio, K. (1996) Hepatology 24, 636-642 Ishiki, Y., Ohnishi, H., Muto, Y., Matsumoto, K., and Nakamura, T. (1992) Hepatology 16, 1227-1235 Ishii, T., Sato, M., Sudo, K., Suzuki, M., Nakai, H., Hishida, T., Niwa, T., Umezu, K., and Yuasa, S. (1995) Journal of biochemistry 117, 1105-1112 Miyazawa, K., Shimomura, T., and Kitamura, N. (1996) J Biol Chem 271, 3615-3618 P., R. J. S. B. D. (2011) UCSD Molecule Pages Vitale, A., and Denecke, J. (1999) The Plant Cell Online 11, 615-628 Horiguchi, K., Hirano, T., Ueki, T., Hirakawa, K., and Fujimoto, J. (2009) Journal of hepato-biliary-pancreatic surgery 16, 171-177 Li, F., Sun, J. Y., Wang, J. Y., Du, S. L., Lu, W. Y., Liu, M., Xie, C., and Shi, J. Y. (2008) Journal of controlled release : official journal of the Controlled Release Society 131, 77-82 Inoue, T., Okada, H., Kobayashi, T., Watanabe, Y., Kanno, Y., Kopp, J. B., Nishida, T., Takigawa, M., Ueno, M., Nakamura, T., and Suzuki, H. (2003) FASEB J 17, 268-270 Kim, W. H., Matsumoto, K., Bessho, K., and Nakamura, T. (2005) Am J Pathol 166, 1017-1028 Taniyama, Y., Morishita, R., Aoki, M., Hiraoka, K., Yamasaki, K., Hashiya, N., Matsumoto, K., Nakamura, T., Kaneda, Y., and Ogihara, T. (2002) Hypertension 40, 47-53 Nishino, M., Iimuro, Y., Ueki, T., Hirano, T., and Fujimoto, J. (2008) Surgery 144, 374-384 Azuma, J., Taniyama, Y., Takeya, Y., Iekushi, K., Aoki, M., Dosaka, N., Matsumoto, K., Nakamura, T., Ogihara, T., and Morishita, R. (2006) Gene Ther 13, 1206-1213 Jiang, D., Jiang, Z., Han, F., Zhang, Y., and Li, Z. (2008) Eur J Appl Physiol 103, 489-493 Kanemura, H., Iimuro, Y., Takeuchi, M., Ueki, T., Hirano, T., Horiguchi, K., Asano, Y., and Fujimoto, J. (2008) Hepatol Res 38, 930-939 Xia, J. L., Dai, C., Michalopoulos, G. K., and Liu, Y. (2006) Am J Pathol 168, 1500-1512 Friedman, S. L. (2008) Nat Biotechnol 26, 399-400 Iimuro, Y., and Brenner, D. A. (2008) Pharm Res 25, 249-258 Siller-Lopez, F., Sandoval, A., Salgado, S., Salazar, A., Bueno, M., Garcia, J., Vera, J., Galvez, J., Hernandez, I., Ramos, M., Aguilar-Cordova, E., and Armendariz-Borunda, J. (2004) Gastroenterology 126, 1122-1133; discussion 1949 Bueno, M., Salgado, S., Beas-Zarate, C., and Armendariz-Borunda, J. (2006) J Gene Med 8, 1291-1299 Roderfeld, M., Weiskirchen, R., Wagner, S., Berres, M. L., Henkel, C., Grotzinger, J., Gressner, A. M., Matern, S., and Roeb, E. (2006) FASEB J 20, 444-454 109   86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109.   Jiang, W., Wang, J. Y., Yang, C. Q., Liu, W. B., Wang, Y. Q., and He, B. M. (2005) Chin Med J (Engl) 118, 192-197 Hu, Y. B., Li, D. G., and Lu, H. M. (2007) J Gene Med 9, 217-229 Qi, Z., Atsuchi, N., Ooshima, A., Takeshita, A., and Ueno, H. (1999) Proc Natl Acad Sci U S A 96, 2345-2349 Arias, M., Sauer-Lehnen, S., Treptau, J., Janoschek, N., Theuerkauf, I., Buettner, R., Gressner, A. M., and Weiskirchen, R. (2003) BMC Gastroenterol 3, 29 Kinoshita, K., Iimuro, Y., Otogawa, K., Saika, S., Inagaki, Y., Nakajima, Y., Kawada, N., Fujimoto, J., Friedman, S. L., and Ikeda, K. (2007) Gut 56, 706714 Chen, S. W., Zhang, X. R., Wang, C. Z., Chen, W. Z., Xie, W. F., and Chen, Y. X. (2008) Liver Int 28, 1446-1457 George, J., and Tsutsumi, M. (2007) Gene Ther 14, 790-803 Ghiassi-Nejad, Z., and Friedman, S. L. (2008) Expert review of gastroenterology & hepatology 2, 803-816 Stone, D., Liu, Y., Shayakhmetov, D., Li, Z. Y., Ni, S., and Lieber, A. (2007) J Virol 81, 4866-4871 Snoeys, J., Lievens, J., Wisse, E., Jacobs, F., Duimel, H., Collen, D., Frederik, P., and De Geest, B. (2007) Gene Ther 14, 604-612 Leopold, P. L., and Crystal, R. G. (2007) Adv Drug Deliv Rev 59, 810-821 Boussif, O., Lezoualc'h, F., Zanta, M. A., Mergny, M. D., Scherman, D., Demeneix, B., and Behr, J. P. (1995) Proc Natl Acad Sci U S A 92, 7297-7301 Habibullah, C. M., Syed, I. H., Qamar, A., and Taher-Uz, Z. (1994) Transplantation 58, 951-952 Blei, A. T. (2005) Liver transplantation : official publication of the American Association for the Study of Liver Diseases and the International Liver Transplantation Society, S30-34 Dai, H., Jiang, X., Leong, K. W., and Mao, H. Q. (2011) Human gene therapy 22, 873-878 Otsuka, M., Baru, M., Delriviere, L., Talpe, S., Nur, I., and Gianello, P. (2000) Journal of drug targeting 8, 267-279 Kuriyama, S., Yoshiji, H., Nakai, S., Deguchi, A., Uchida, N., Kimura, Y., Inoue, H., Kinekawa, F., Ogawa, M., Nonomura, T., Masaki, T., Kurokohchi, K., and Watanabe, S. (2005) Oncology reports 13, 69-74 Beljaars, L., Molema, G., Weert, B., Bonnema, H., Olinga, P., Groothuis, G. M., Meijer, D. K., and Poelstra, K. (1999) Hepatology 29, 1486-1493 Beljaars, L., Olinga, P., Molema, G., de Bleser, P., Geerts, A., Groothuis, G. M., Meijer, D. K., and Poelstra, K. (2001) Liver 21, 320-328 Beljaars, L., Molema, G., Schuppan, D., Geerts, A., De Bleser, P. J., Weert, B., Meijer, D. K., and Poelstra, K. (2000) J Biol Chem 275, 12743-12751 Sato, Y., Murase, K., Kato, J., Kobune, M., Sato, T., Kawano, Y., Takimoto, R., Takada, K., Miyanishi, K., Matsunaga, T., Takayama, T., and Niitsu, Y. (2008) Nat Biotechnol 26, 431-442 Iredale, J. P. (2001) Semin Liver Dis 21, 427-436 Blouin, A., Bolender, R. P., and Weibel, E. R. (1977) J Cell Biol 72, 441-455 Tilg, H., and Diehl, A. M. (2000) N Engl J Med 343, 1467-1476 110   110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124. 125. 126. 127. 128. 129. 130. 131. 132. 133. 134. 135. 136. 137.   Guyot, C., Lepreux, S., Combe, C., Doudnikoff, E., Bioulac-Sage, P., Balabaud, C., and Desmouliere, A. (2006) Int J Biochem Cell Biol 38, 135151 Dooley, S., Delvoux, B., Lahme, B., Mangasser-Stephan, K., and Gressner, A. M. (2000) Hepatology 31, 1094-1106 Border, W. A., and Noble, N. A. (1994) N Engl J Med 331, 1286-1292 Franklin, T. J. (1997) Int J Biochem Cell Biol 29, 79-89 Stella, M. C., and Comoglio, P. M. (1999) Int J Biochem Cell Biol 31, 13571362 Inoue, H., Yokoyama, F., Kita, Y., Yoshiji, H., Tsujimoto, T., Deguchi, A., Nakai, S., Morishita, A., Uchida, N., Masaki, T., Watanabe, S., and Kuriyama, S. (2006) Int J Mol Med 17, 857-864 Florquin, S., and Rouschop, K. M. (2003) Kidney Int Suppl, S15-20 Munger, J. S., Harpel, J. G., Gleizes, P. E., Mazzieri, R., Nunes, I., and Rifkin, D. B. (1997) Kidney Int 51, 1376-1382 Wipff, P. J., and Hinz, B. (2008) Eur J Cell Biol 87, 601-615 Kondou, H., Mushiake, S., Etani, Y., Miyoshi, Y., Michigami, T., and Ozono, K. (2003) J Hepatol 39, 742-748 Breitkopf, K., Sawitza, I., Westhoff, J. H., Wickert, L., Dooley, S., and Gressner, A. M. (2005) Gut 54, 673-681 Hayashi, H., Sakai, K., Baba, H., and Sakai, T. (2012) Hepatology 55, 15621573 Scarpino, S., Di Napoli, A., Taraboletti, G., Cancrini, A., and Ruco, L. P. (2005) J Pathol 205, 50-56 Bezerra, J. A., Bugge, T. H., Melin-Aldana, H., Sabla, G., Kombrinck, K. W., Witte, D. P., and Degen, J. L. (1999) Proc Natl Acad Sci U S A 96, 1514315148 Waisman, D. M. (2003) Plasminogen: Structure, Activation and Regulation, Wang, H., Zhang, Y., and Heuckeroth, R. O. (2007) FEBS Lett 581, 30983104 Martinez-Rizo, A., Bueno-Topete, M., Gonzalez-Cuevas, J., and ArmendarizBorunda, J. (2010) Liver Int 30, 298-310 Ghosh, A. K., and Vaughan, D. E. (2012) J Cell Physiol 227, 493-507 Hu, P. F., Chen, H., Zhong, W., Lin, Y., Zhang, X., Chen, Y. X., and Xie, W. F. (2009) J Hepatol 51, 102-113 Pedrozo, H. A., Schwartz, Z., Robinson, M., Gomes, R., Dean, D. D., Bonewald, L. F., and Boyan, B. D. (1999) Endocrinology 140, 5806-5816 Zheng, G., and Harris, D. C. (2004) Kidney Int 66, 455-456 Hughes, R. D., Mitry, R. R., and Dhawan, A. (2012) Transplantation 93, 342347 310.1097/TP.1090b1013e31823b31872d31826 Ochiya, T., Yamamoto, Y., and Banas, A. (2010) Differentiation 79, 65-73 Seglen, P. O. (1976) Methods Cell Biol 13, 29-83 Vogel, S., Piantedosi, R., Frank, J., Lalazar, A., Rockey, D. C., Friedman, S. L., and Blaner, W. S. (2000) J Lipid Res 41, 882-893 Sato, Y., and Rifkin, D. B. (1989) J Cell Biol 109, 309-315 Longstaff, C. (1994) Blood Coagul Fibrinolysis 5, 537-542 Budinger, G. R., Mutlu, G. M., Eisenbart, J., Fuller, A. C., Bellmeyer, A. A., Baker, C. M., Wilson, M., Ridge, K., Barrett, T. A., Lee, V. Y., and Chandel, N. S. (2006) Proc Natl Acad Sci U S A 103, 4604-4609 111   138. 139. 140. 141. 142. 143. 144. 145. 146. 147. 148. 149. 150. 151. 152. 153. 154. 155. 156. 157. 158. 159. 160. 161. 162.   van't Veer, M. B., Brooijmans, A. M., Langerak, A. W., Verhaaf, B., Goudswaard, C. S., Graveland, W. J., van Lom, K., and Valk, P. J. (2006) Haematologica 91, 56-63 Bhandari, R. N., Riccalton, L. A., Lewis, A. L., Fry, J. R., Hammond, A. H., Tendler, S. J., and Shakesheff, K. M. (2001) Tissue Eng 7, 345-357 Abu-Absi, S. F., Hansen, L. K., and Hu, W. S. (2004) Cytotechnology 45, 125140 Murphy-Ullrich, J. E., Schultz-Cherry, S., and Hook, M. (1992) Mol Biol Cell 3, 181-188 Nishimura, K., Matsumiya, K., Miura, H., Tsujimura, A., Nonomura, N., Matsumoto, K., Nakamura, T., and Okuyama, A. (2003) Int J Androl 26, 175179 Yang, J., Dai, C., and Liu, Y. (2005) J Am Soc Nephrol 16, 68-78 Anonick, P. K., Yoo, J. K., Webb, D. J., and Gonias, S. L. (1993) Biochem J 289 ( Pt 3), 903-909 Bonnefoy, A., and Legrand, C. (2000) Thromb Res 98, 323-332 Hogg, P. J., Stenflo, J., and Mosher, D. F. (1992) Biochemistry 31, 265-269 Olsen, A. L., Bloomer, S. A., Chan, E. P., Gaca, M. D., Georges, P. C., Sackey, B., Uemura, M., Janmey, P. A., and Wells, R. G. (2011) Am J Physiol Gastrointest Liver Physiol 301, G110-118 Scotton, C. J., and Chambers, R. C. (2007) Chest 132, 1311-1321 Shek, F. W., and Benyon, R. C. (2004) Eur J Gastroenterol Hepatol 16, 123126 Schuppan, D., and Pinzani, M. (2012) J Hepatol 56 Suppl 1, S66-74 Fallowfield, J. A. (2011) Am J Physiol Gastrointest Liver Physiol 300, G709715 Poelstra, K., and Schuppan, D. (2011) J Hepatol 55, 726-728 Hu, Z., Evarts, R. P., Fujio, K., Marsden, E. R., and Thorgeirsson, S. S. (1993) Am J Pathol 142, 1823-1830 Ebrahimkhani, M. R., Oakley, F., Murphy, L. B., Mann, J., Moles, A., Perugorria, M. J., Ellis, E., Lakey, A. F., Burt, A. D., Douglass, A., Wright, M. C., White, S. A., Jaffre, F., Maroteaux, L., and Mann, D. A. (2011) Nat Med 17, 1668-1673 Hirata, K., Ogata, I., Ohta, Y., and Fujiwara, K. (1989) J Pathol 158, 157-165 Wang, Z. X., Wang, Z. G., Ran, H. T., Ren, J. L., Zhang, Y., Li, Q., Zhu, Y. F., and Ao, M. (2009) Clin Imaging 33, 454-461 Tabata, Y., and Ikada, Y. (1988) Biomaterials 9, 356-362 Chouly, C., Pouliquen, D., Lucet, I., Jeune, J., and Jallet, P. (1996) Journal of microencapsulation 13, 245-255 Jiang, X., Dai, H., Ke, C. Y., Mo, X., Torbenson, M. S., Li, Z., and Mao, H. Q. (2007) Journal of controlled release : official journal of the Controlled Release Society 122, 297-304 Tai, D. C., Tan, N., Xu, S., Kang, C. H., Chia, S. M., Cheng, C. L., Wee, A., Wei, C. L., Raja, A. M., Xiao, G., Chang, S., Rajapakse, J. C., So, P. T., Tang, H. H., Chen, C. S., and Yu, H. (2009) J Biomed Opt 14, 044013 Wisse, E., Braet, F., Duimel, H., Vreuls, C., Koek, G., Olde Damink, S. W., van den Broek, M. A., De Geest, B., Dejong, C. H., Tateno, C., and Frederik, P. (2010) World J Gastroenterol 16, 2851-2866 Martinez-Hernandez, A., and Martinez, J. (1991) Hepatology 14, 864-874 112   163. 164. 165. 166. 167. 168. 169. 170. 171. 172. 173. 174. 175. 176. 177. 178. 179. 180. 181. 182. 183. 184. 185. 186.   Schaffner, F., and Poper, H. (1963) Gastroenterology 44, 239-242 DeLeve, L. D. (2007) Semin Liver Dis 27, 390-400 Anderson, N., and Borlak, J. (2008) Mechanisms of Toxic Liver Injury. in Hepatotoxicity: From Genomics to In Vitro and In Vivo Models (Sahu, S. C. ed.), John Wiley & Sons. pp 191-286 Heinloth, A. N., Irwin, R. D., Boorman, G. A., Nettesheim, P., Fannin, R. D., Sieber, S. O., Snell, M. L., Tucker, C. J., Li, L., Travlos, G. S., Vansant, G., Blackshear, P. E., Tennant, R. W., Cunningham, M. L., and Paules, R. S. (2004) Toxicol Sci 80, 193-202 Matsuzaki, S., Onda, M., Tajiri, T., and Kim, D. Y. (1997) Hepatology 25, 828-832 Iredale, J. (2008) Clin Med 8, 29-31 Adrian, J. E., Poelstra, K., Scherphof, G. L., Meijer, D. K. F., van LoenenWeemaes, A.-m., Reker-Smit, C., Morselt, H. W. M., Zwiers, P., and Kamps, J. A. A. M. (2007) Journal of Pharmacology and Experimental Therapeutics 321, 536-543 Son, G., Hines, I. N., Lindquist, J., Schrum, L. W., and Rippe, R. A. (2009) Hepatology 50, 1512-1523 Xue, F., Takahara, T., Yata, Y., Kuwabara, Y., Shinno, E., Nonome, K., Minemura, M., Takahara, S., Li, X., Yamato, E., and Watanabe, A. (2003) Gut 52, 694-700 Dai, H., Jiang, X., Tan, G. C., Chen, Y., Torbenson, M., Leong, K. W., and Mao, H. Q. (2006) Int J Nanomedicine 1, 507-522 Otsuka, M., Baru, M., Delrivière, L., Talpe, S., Nur, I., and Gianello, P. (2000) Journal of drug targeting 8, 267-279 Derynck, R., Chen, R. H., Ebner, R., Filvaroff, E. H., and Lawler, S. (1994) Princess Takamatsu Symp 24, 264-275 Massague, J., and Weis-Garcia, F. (1996) Cancer Surv 27, 41-64 Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465-471 Sun, Y., Liu, X., Ng-Eaton, E., Lodish, H. F., and Weinberg, R. A. (1999) Proc Natl Acad Sci U S A 96, 12442-12447 Esposito, C., Parrilla, B., Cornacchia, F., Grosjean, F., Mangione, F., Serpieri, N., Valentino, R., Villa, L., Arra, M., Esposito, V., and Dal Canton, A. (2009) Growth Factors 27, 173-180 Liu, Y. (2004) Am J Physiol Renal Physiol 287, F7-16 Soto-Gutierrez, A., Navarro-Alvarez, N., Yagi, H., and Yarmush, M. L. (2009) Curr Opin Organ Transplant 14, 667-673 Fitzpatrick, E., Mitry, R. R., and Dhawan, A. (2009) J Intern Med 266, 339357 Dhawan, A., Mitry, R. R., and Hughes, R. D. (2006) J Inherit Metab Dis 29, 431-435 Bumgardner, G. L., and Orosz, C. G. (2000) Immunol Rev 174, 260-279 Takahashi, K., and Yamanaka, S. (2006) Cell 126, 663-676 Nakagawa, M., Koyanagi, M., Tanabe, K., Takahashi, K., Ichisaka, T., Aoi, T., Okita, K., Mochiduki, Y., Takizawa, N., and Yamanaka, S. (2008) Nat Biotech 26, 101-106 Roelandt, P., Pauwelyn, K. A., Sancho-Bru, P., Subramanian, K., Bose, B., Ordovas, L., Vanuytsel, K., Geraerts, M., Firpo, M., De Vos, R., Fevery, J., Nevens, F., Hu, W. S., and Verfaillie, C. M. (2010) PLoS One 5, e12101 113   187.   Logan, C. Y., and Nusse, R. (2004) Annual Review of Cell and Developmental Biology 20, 781-810 114   APPENDICES a APPENDIX 1: PUBLICATIONS 1. BC Narmada*, Y Kang*, L Venkatraman, Q Peng, RB Sakban, B Nugraha, X Jiang, RM Bunte, PTC So, L Tucker-Kellogg, HQ Mao & H Yu. HSC-targeted delivery of HGF transgene via bile duct infusion enhances its expression at fibrotic foci to regress DMN-induced liver fibrosis. Human Gene Therapy. August 2012 (Submitted) 2. SM Chia*, L Venkatraman*, N Tan, S Chang, KF Yee, BC Narmada, JJ Wang, CH Kang, SS Bhowmick, P So, L Tucker-Kellogg and H Yu. Plasmin and Thrombospondin Interplay in TGF-β1 Activation Regulates Liver Fibrosis and Regression. J Cellular Physiology. July 2012 (Submitted). 3. L Venkatraman, SM Chia, BC Narmada, SS Bhowmick, CF Dewey Jr., JK White, L Tucker-Kellogg and H Yu. Plasmin Triggers a Switch-like Decrease in Thrombospondin-Dependent Activation of TGF-β1. Biophysical Journal. July 2012 (Accepted) 4. BC Narmada, SM Chia, L Tucker-Kellogg and H Yu. HGF regulates the activation of TGF- β1 in rat hepatocytes and hepatic stellate cells. J Cellular Physiology. June 2012. 5. A Ananthanarayanan, L Tucker-Kellogg, BC Narmada, L Venkatraman, N Rahim, Y Wang, KC Huen, and H Yu. Systems Biology in Biomaterials and Tissue Engineering. In Book: Comprehensive Biomaterials. P Ducheyne, KE Healy, D Hutmacher, DE Grainger & CJ Kirkpatrick. August 2011 6. A Ananthanarayanan, BC Narmada, M Xuejun and H Yu. Purpose Driven Biomaterials in Liver Tissue Engineering. Trends in Biotechnology. March 2011. 29(3):110-8 7. W Zhang*, L Tucker-Kellogg*, BC Narmada, L Venkatraman, S Chang, Y Lu, N Tan, JK White, RR Jia, SS Bhowmick, S Shen, CF Dewey Jr. and H Yu. Cell Delivery Therapeutics for Liver Regeneration. Adv Drug Delivery Rev. June 2010. 62(7-8):814-26 b APPENDIX 2: CONFERENCE PROCEEDINGS 1. Keystone Symposia meeting on Fibrosis: Translation of Basic Research to Human Disease and Novel Therapeutics, March-April 2012, Big Sky, Montana USA (Poster Presentation) 2. TERMIS-AP meeting, August 2011, Singapore (Oral Presentation) 3. Keystone Symposia meeting on TGF-beta in Immune Responses: From Bench to Bedside, January 2011, Snowbird, Utah USA (Poster Presentation) 4. GPBE/NUS-Tohoku Conference on Bioengineering, May 2008, Sendai, Japan (Oral Presentation) 5. GPBE/NUS-Tohoku Graduate Student Conference in Bioengineering, December 2008, NUS, Singapore (Poster Presentation) c [...]... markers by the hepatocyte-like cells derived from hiPSCs xv CHAPTER 1   1   1 INTRODUCTION 1. 1 LIVER STRUCTURE AND FUNCTION 1. 1 .1 Anatomy Of The Liver Liver is the body’s largest internal organ and is responsible for a wide spectrum of functions Its strategic location at the interface of the digestive system is crucial in the processing of absorbed nutrients through the metabolism of glucose, proteins... non-parenchymal cells of the liver, the HSCs play an important role and their interaction with hepatocytes constitutes the regulation of hepatocyte functions The HSCs constitute of 1. 5% of the liver volume and constitutes up to 8% of the liver cells HSCs were first identified and classified as fat-storing cells containing lipid droplets by Ito in 19 52 and therefore initially referred to as Ito cells, well... Activation of hepatic stellate cells (HSCs) leads to overproduction of transforming growth factor-beta 1 (TGF- 1) , the key cytokine involved in fibrogenesis and collagen accumulation Concentrated regions with increasing numbers of activated HSCs lead to the formation of α-SMA & collagen-rich fibrotic foci that lead to the deregulation of liver homeostasis resulting in fibrosis Hepatocyte growth factor (HGF)... liver injury For example, residual mature hepatocytes and cholangiocytes proliferate to restore liver mass after acute partial hepatectomy, while liver progenitors are involved in the repair of chronically injured livers, or in other special kind of injuries 1. 1.3 .1. 1 Hepatocyte functions Healthy functional hepatocyte cultures are an absolute essential for drug toxicity screening, drug metabolism studies... for the treatment of liver fibrosis Better understanding of the mechanism of action of HGF and development of a robust delivery method that can address the risk of hepatocarcinogenesis from untargeted HGF therapy are quintessential towards the development of HGF-based anti-fibrotic therapies To gain insight into the anti-fibrotic role of HGF, we studied the effects of HGF on the TGF- 1 activation pathway... PAI -1 plasminogen activator inhibitor – 1 PCR polymerase chain reaction PDGF platelet-derived growth factor PECAM -1 platelet endothelial cell adhesion molecule -1 PLG plasminogen PPAR peroxisome proliferator-activated receptor SECs sinusoidal endothelial cells SHG second harmonic generation TAA thioacetamide TGF- 1 transforming growth factor – beta 1 TIMP -1 tissue inhibitors of metalloproteinase -1 TIMP-2... and cytokines released from activated Kupffer cells (25,26)   13   Liver fibrosis results from continuous injury to the liver, including viral hepatitis, alcohol abuse, metabolic diseases, autoimmune diseases, and cholestatic liver diseases In other words, fibrosis is a consequence of the excessive healing response triggered by chronic liver injury The end stage of liver fibrosis, cirrhosis, is histologically... response to variety fibrogenic stimuli According to the most recent studies, the major sources of hepatic myofibroblasts in experimental liver fibrosis are hepatic stellate cells and portal fibroblasts (35) 16   1. 2.2 .1 Hepatic Fibrosis And Myofibroblasts The postulate that HSCs may be the precursors of the cells responsible for excessive synthesis of ECM and hepatic fibrosis was confirmed in a number of. .. electron micrographs of hepatic sinusoids in processed liver tissues collected from DMN-induced fibrotic rats at 4th week 1. 2.2.2 Activation Of HSCs Into Myofibroblasts Activation of HSCs is a primary tissue repair response to hepatic injury of various types The process of activation or transdifferentiation of HSCs into myofibroblast17   like phenotype (Fig 8) includes progressive loss of retinoids, proliferation,... retinoids, proliferation, expression of alpha-smooth muscle actin (α-SMA), increased contractility, and enhanced production of inflammatory cytokines and ECM proteins (24) Activated HSCs are considered to be the major cell type to deposit ECM and perpetuate fibrosis of the liver A large body of evidence with regard to the mediators and mechanisms of activation and proliferation of HSCs is obtained from in vitro . HOMEOSTATIC REGULATION OF CYTOKINES TO RETARD LIVER FIBROSIS BALAKRISHNAN CHAKRPANI NARMADA NATIONAL UNIVERSITY OF SINGAPORE 2 012 HOMEOSTATIC REGULATION OF CYTOKINES TO RETARD LIVER FIBROSIS. FUNCTION……………………………… .1 1 .1. 1. Anatomy Of The Liver ………………………………………… 1 1. 1.2. Liver Functions And The Importance of Zonation………………… 4 1. 1.3. Cells In The Sinusoidal Lumen…………………………………… 5 1. 1.3 .1. Hepatocytes………………………………………………………5. 1. 1.3.4 .1. The functional role of liver sinusoidal endothelia……… .12 1. 2. FIBROSIS ……………………………………………………………… .13 1. 2 .1. Etiology…………………………………………………………… 15 1. 2.2. Homeostatic Regulation Of Cytokines