Dynamics of Endogenous Cardiac Repair and Methods for Enhanced Post-Injury Cell Therapy

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Doctor of Philosophy (PhD)
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stem cells
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Heart attacks are a leading cause of mortality in the United States, responsible for over 500,000 deaths annually. Despite advancing treatments for acute heart attack, 5-year mortality exceeds 50% as the organ fails to heal the resulting scar. Recent studies revealed modest cardiac regeneration occurring throughout life and accelerating (albeit insufficiently) post-injury. However, the magnitude is contested with some studies indicating low cardiomyocyte formation and others indicating rapid formation of increasingly inferior cardiomyocytes. Resolving this question determines the needed strategy for repair augmentation. Chapter 3 scrutinizes current apparently-paradoxical studies and offers a unified estimate of cardiomyocyte turnover via a hybrid-model software platform. As limited engraftment (<2%) was cited as a primary impediment in bone marrow cell (BMC) infusion clinical trials, Chapter 4 recapitulates these trials in an intact-organ murine model--the isolated perfused heart. Flow cytometry enables objective, sensitive identification of strongly-retained BMC phenotypes. Results show that endothelial P-selectin surfaces at 30 minutes post ischemia-reperfusion injury, leading to preferential engraftment of c-kit+ BMCs (which exhibit superior P-selectin adherence in vitro). Chapter 5 adapts the flow cytometry technique to measurement of absolute cell retention (non-ratiometric) to evaluate chemotactic properties of Stromal Derived Factor (SDF)-eluting implants of polymerized hyaluronic acid. Stem cells home to chemokine concentration gradients and thus SDF-eluting hydrogels can draw infused stem cells to the implant site. The hydrogel increases cardiac BMC homing by 5-fold, confirming that local chemokine milieu alteration can augment BMC therapy. Leveraging Chapter 4 results, Chapter 6 artificially stimulates P-selectin endothelialization in quiescent endothelium to improve BMC engraftment rates even after endogenous activation subsides. Low-dose peroxide, a reactive oxygen species known to induce brief inflammation, when delivered prior to BMC infusion, enhances retention by 3-fold. Interestingly, peroxide-induced c-kit+ BMC retention rates are equivalent to true ischemic injury rates, while c-kit-negative BMCs also experience enhanced engraftment. This work spans the scientific process, conducting basic research of natural physiology and leveraging results to propose and test two promising therapeutic strategies--alteration of local chemokine concentrations and endothelial adhesion molecule display. Additionally, new techniques, including computational methods and flow cytometry-based engraftment assays enable future work in cardiac regeneration.

Kenneth B. Margulies
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