Acute myocardial infarction (AMI) rat model was adopted to test the effect of AngioT in vivo. AngioT was directly injected into the ischaemic region of the left ventricle immediately after the ligation of left anterior descending artery. The densities of vessels in AngioT treated hearts were on average 3-4 folds higher compared with non-treated hearts after two and seven days post infarction. Using TUNEL method, approximately 3-fold lower numbers of apoptotic cardiomyocytes were detected in AngioT treated AMI hearts compared with controls. The infarcted volume estimated by Masson's Trichrome staining was significantly decreased in AngioT treated hearts (27.44%+/-7.34% vs. 39.53%+/-5.97%, p<0.05) compared with control hearts 14 days post infarction. Echocardiography demonstrated that left ventricular ejection fraction and fraction shortening in AngioT treated hearts were significantly improved by 10.4% and 22.3% compared to those in the control hearts 2-day post infarction (p<0.05). These improvements were maintained for 2-week post infarction. / Based on the analysis of rat angiogenesis specific superarray, VEGFb, VEGFc and FGF7, were found to be highly expressed in AngioT treated AMI hearts compared to the controls. The expression levels of survival related genes Bcl2 and Akt1 were increased to 3.3 and 2.8 folds respectively in AngioT treated AMI hearts compared with the controls (both p<0.05). Based on the signal transduction pathway finder superarray, Jak-Stat pathway activators, Interleukin 4 receptor and Interferon regulatory factor 1 (IL4R and IRF1), were found to be highly expressed in the AngioT treated AMI heart. / In conclusion, bioactive angiogenic factors (AngioT) were isolated from Geum. japonicum Thunb. Var. Chinense F. Bolle (GJ). Intra-cardiomuscular injection of AngioT had beneficial effects on the acute myocardial infarction. The underlying mechanisms might be related to the activation of Jak-Stat Pathway and over expressions of angiogenic factors and survival associated genes. The therapeutic properties of AngioT appear entirely novel and may provide a new dimension for therapeutic angiogenesis for the treatment of acute ischaemic heart disease. / In the present study, an angiogenic tannins fraction (AngioT) was isolated from Geum. japonicum Thunb. Var. Chinense F. Bolle using bio-assay guided strategy. AngioT increased the proliferation of human umbilical vein endothelial cell (HUVEC) in culture within 24-hour, 48-hour and 72-hour treatment in a dose-dependent pattern. The EC50 of AngioT on HUVEC was less than 25mug/ml. Conditional media from AngioT treated HUVEC stimulated the proliferation of HUVEC significantly greater than conditional media from non-treated HUVEC. Using 2-dimensional electrophoresis and MALDI-TOF, VEGFa was identified in the AngioT treated conditional medium. / Ischaemic heart disease remains the leading cause of morbidity and mortality in most countries. Severe ischaemia of myocardium induces myocardial infarction and results in an irreversible loss of myocardium. Restoration of coronary blood flow by rapid angiogenesis may offer a direct and effective therapeutic way to intractable ischaemic heart diseases. / Key words. ischaemic heart disease; myocardial infarction; therapeutic angiogenesis; Geum japonicum; apoptosis; tannins; VEGF; Jak-Stat pathway / Gu, Xuemei. / "Sep 2007." / Adviser: Peter Tong Chun Yip. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4615. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 159-177). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Heart failure, as a result of myocardial infarction, is a major cause of mortality in human. The main cause of heart failure is that when adult cardiomyocytes die in the infarct site they do not regenerate. Instead the infract site is replaced by fibroblasts and collagen scar. It is generally believed that cardiomyocytes have terminally differentiated and can not divide to replace cardiomyocytes that have lost following injury. However, recently published data have provided new evidence that there is a small but continuously turnover of cardiomyocytes in the adult heart. These new findings provide a new theory that the heart does possess a limited ability to regenerate. / I also examined the regenerative ability of cardiomyocyte in adult heart. MRL mice were used because previously it has been reported that the cardiomyocyte could proliferate in response to injury. To understand how the cardiomyocytes in the MRL mouse heart, I used a cryo-injury approach. I discovered that the cardiomyoctyes in MRL mouse hearts were capable of dividing shortly after cryo-injury. These MRL hearts healed without scarring in contrast to C57BL/6 control mice. It was discovered that BMP-2, GATA4 and Nkx2.5 were involved in the healing process. The activation of these genes induced the cardiomyocyte to re-enter the cell cycle so that new cardiomyocytes could replace the cell that have been lost in the infarct site. I also discovered that stem cells may also play a minor role in the healing process. / In summary, my research findings revealed that cardiomyocytes regeneration in the heart is a very complex process that involves the participation of many cells and signalling pathway. There findings raise many intriguing and important questions and are worthy of being addressed in the future. / Stem cell therapy has been proposed as a potential treatment for various myocardial diseases. Chen et al. (2004) found small chemical called reversine that could dedifferentiate C2C12 cells to become stem-like cells. In this study, I demonstrated that reversine could inhibit the growth of C2C12 cell. The presence of reversine in cell culture could significantly inhibit muscle-specific genes MyoD, Myogenin and Myf5 expression. These 3 muscle specific transcriptional genes are essential for maintaining muscle differentiation. The down regulation of these gene showed that reversine could dedifferentiate C2C12 cells. We also discovered that reversine-treated C2C12 cells could differentiate into cardiomyocytes when they were cocultured with cardiomyocytes or when transplanted into the infarct site of a cryo-injured heart. / To investigate the regenerative potential of cardiomyoctyes in adult heart, we tried first to uncover the signals that direct post-natal cardiomyocytes to enter into growth arrest and differentiation. In the first part of my study, I established that the cardiomycytes divided extensively in 2 day-old post-natal hearts and that the majority of these cells entered into growth arrest and terminal differentiation at day 13. Comparative proteomic techniques were used in order to identify proteins that might be associated with cardiomyocytes proliferation during terminal differentiation the mouse heart. Several proteins were found to be differently expressed and amongst them was cyclin I protein. Cyclin I was found strongly expressed in 13 day old hearts. The protein is involved in signaling growth arrested in cells. / Liu, Ye. / "November 2006." / Adviser: Lee Ka Ho. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5658. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 142-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Leung, Sze Lee Cecilia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 144-153). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.iii / Acknowledgements --- p.v / Table of Content --- p.vi / Abbreviations --- p.xv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Stem cells --- p.1 / Chapter 1.1.1 --- Adult stem cells --- p.2 / Chapter 1.1.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5 / Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6 / Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7 / Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7 / Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9 / Chapter 1.2.1 --- Causes and statistics of CVD --- p.9 / Chapter 1.2.2 --- Current treatment for CVD --- p.10 / Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11 / Chapter 1.3 --- Myosin light chain2v --- p.13 / Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14 / Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15 / Chapter 1.6 --- Aims of the Project --- p.19 / Chapter 1.7 --- Significance of the Project --- p.19 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Subcloning --- p.20 / Chapter 2.1.1 --- Amplification of MLC-2v --- p.20 / Chapter 2.1.2 --- Purification of DNA product --- p.21 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.21 / Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22 / Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22 / Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23 / Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23 / Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24 / Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25 / Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25 / Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26 / Chapter 2.2.1 --- Derivation of MEF --- p.26 / Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27 / Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28 / Chapter 2.3 --- HESC culture --- p.29 / Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29 / Chapter 2.3.2 --- Splitting hESCs --- p.30 / Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31 / Chapter a) --- Distinguish differentiated and undifferentiated cells and colonies / Chapter b) --- "Remove differentiated cells by ""Picking to Remove""" / Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep""" / Chapter 2.3.4 --- Freezing hESCs --- p.31 / Chapter 2.3.5 --- Differentiation of hESCs --- p.32 / Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34 / Chapter a) --- Preparation of mTeSRTMl / Chapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquots / Chapter c) --- Coating plates with BD MatrigelTM hESC-qualified Matrix / Chapter d) --- Human Embryonic stem cells culture in mTeSRTMl / Chapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36 / Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36 / Chapter 2.4.2 --- Immunofluorescence staining --- p.37 / Chapter 2.5 --- MESC culture --- p.38 / Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38 / Chapter 2.5.2 --- Splitting mESCs --- p.38 / Chapter 2.5.3 --- Differentiation of mESCs --- p.39 / Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40 / Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41 / Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41 / Chapter 2.6.2 --- LV titering --- p.42 / Chapter 2.7 --- MultipleTransduction --- p.43 / Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43 / Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43 / Chapter 2.8.2 --- Selection of stable clones --- p.44 / Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45 / Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45 / Chapter 2.9.2 --- FACS --- p.46 / Chapter 2.10 --- Gene expression study / Chapter 2.10.1 --- Primer design --- p.46 / Chapter 2.10.2 --- RNA extraction --- p.46 / Chapter 2.10.3 --- DNase Treatment --- p.47 / Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47 / Chapter 2.10.5 --- Quantitative real-time PCR --- p.48 / Chapter 2.10.6 --- Quantification of mRNA expression --- p.49 / Chapter 2.11 --- Protein Expression study --- p.49 / Chapter 2.11.1 --- Crude protein extraction --- p.49 / Chapter 2.11.2 --- Quantitation of protein samples --- p.50 / Chapter 2.11.3 --- SDS-PAGE --- p.50 / Chapter 2.11.4 --- Western Blot --- p.51 / Chapter 2.11.5 --- Western blot luminal detection --- p.52 / Chapter 2.11.6 --- Quantification of protein expression --- p.52 / Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCs / Chapter 3.1 --- Subcloning --- p.57 / Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58 / Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59 / Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60 / Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61 / Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61 / Chapter 3.2 --- Lentivirus (LV) packaging --- p.62 / Chapter 3.2.1 --- Transfection --- p.63 / Chapter 3.2.2 --- LV titering --- p.64 / Chapter 3.3 --- HESC culture --- p.66 / Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67 / Chapter 3.5 --- Differentiation after transduction --- p.69 / Chapter 3.6 --- Antibiotic selection --- p.71 / Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72 / Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72 / Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73 / Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74 / Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75 / Chapter 3.7 --- Differentiation of hESCs after selection --- p.76 / Chapter 3.8 --- FACS --- p.77 / Chapter 3.9 --- QPCR of cells after FACS --- p.80 / Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81 / Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82 / Chapter 3.9.3 --- Gene expression of c-TnT --- p.83 / Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84 / Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATION / Chapter 4.1 --- mESC culture --- p.85 / Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86 / Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88 / Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94 / Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95 / Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98 / Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101 / Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104 / Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107 / Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110 / Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113 / Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114 / Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116 / Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118 / Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120 / Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122 / Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124 / Chapter CHAPTER 5 --- DISCUSSION / Chapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127 / Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135 / Chapter 5.3 --- Conclusion --- p.142 / REFERENCES --- p.144

Thesis (Ph. D.)--Ohio State University, 2007. / Full text release at OhioLINK's ETD Center delayed at author's request

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-176) Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 156-176) Also available online.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 132-147). Also available on the World Wide Web.

Thesis (M.Sc.)--York University, 2008. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 77-94). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:MR51626

Title from first page of PDF document. Includes bibliographical references (p. 19-22).

The ability to generate cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides unprecedented opportunities in the study and treatment of cardiac diseases. The objective of this dissertation is the development of novel methods of utilizing hiPSC-derived cardiomyocytes (hiPSC-CMs). First, we leveraged the potential of hiPSC-CMs to model congenital heart disease caused by mutations in the transcription factor ZIC3. We developed a method to directly explore the effect of ZIC3 inhibition using hiPSCs at a molecular, cellular, and functional level through utilization of CRISPR interference. Our results identified the role of ZIC3 in regulating Nodal signaling, Wnt signaling, and cell structure and motility processes during cardiac development, suggesting that ZIC3 mutation leads to congenital heart disease in humans by the abnormal regulation of multiple steps during left-right axis establishment. Next, we leveraged the potential of hiPSC-CMs to treat myocardial infarction. We demonstrated that the extended delivery of extracellular vesicles secreted by hiPSC-CMs could attenuate injury and promote recovery of the heart after infarction by regulating apoptosis and inflammatory pathways. These results suggest that hiPSC-CM secreted extracellular vesicles represent a novel cell free tool in the treatment of myocardial infarction and the understanding of heart recovery.