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

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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Dynamics of Endogenous Cardiac Repair and Methods for Enhanced Post-Injury Cell Therapy Jeremy Alan Elser University
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University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Dynamics of Endogenous Cardiac Repair and Methods for Enhanced Post-Injury Cell Therapy Jeremy Alan Elser University of Pennsylvania, Follow this and additional works at: Part of the Biomedical Commons Recommended Citation Elser, Jeremy Alan, Dynamics of Endogenous Cardiac Repair and Methods for Enhanced Post-Injury Cell Therapy (2012). Publicly Accessible Penn Dissertations This paper is posted at ScholarlyCommons. For more information, please contact Dynamics of Endogenous Cardiac Repair and Methods for Enhanced Post-Injury Cell Therapy Abstract 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. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) This dissertation is available at ScholarlyCommons: Graduate Group Bioengineering First Advisor Kenneth B. Margulies Keywords cardiac, endothelium, engraftment, regeneration, stem cells, therapy Subject Categories Biomedical This dissertation is available at ScholarlyCommons: DYNAMICS OF ENDOGENOUS CARDIAC REPAIR AND METHODS FOR ENHANCED POST-INJURY CELL THERAPY Jeremy Alan Elser A DISSERTATION in Bioengineering Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2012 Supervisor of Dissertation Dr. Kenneth B Margulies, Professor of Medicine Graduate Group Chairperson Dr. Beth Winkelstein, Professor of Bioengineering Dissertation Committee Dr. Daniel A Hammer (Chair), Professor of Bioengineering Dr. John P Gearhart, Director Institute of Regenerative Medicine Dr. Jason A Burdick, Associate Professor of Bioengineering DYNAMICS OF ENDOGENOUS CARDIAC REPAIR AND METHODS FOR ENHANCED POST-INJURY CELL THERAPY COPYRIGHT 2012 Jeremy Alan Elser This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit Dedication To the giants on whose shoulders we stand iii ACKNOWLEDGMENT I sincerely thank the people who have contributed to this work. In particular, I would like to thank my advisor Dr. Kenneth Margulies, whose love of science maintained my morale, whose professionalism I will always attempt to emulate, and whose keen logical judgment I trust as I trust my own. I would also like to thank Dr. Anbin Mu, our star surgeon, without whom not one of the enclosed experiments could have been performed. Additionally I would like to thank Brendan Purcell, who contributed greatly to Chapter 5 of this manuscript; Brendan was a true brother-in-arms during a full year of tough but ultimately successful collaborative research. I also thank my labmates, all of whom I have tapped at various times for experimental insight, these include Rosa Alvarez, Dr. Abigail Dean, Dr. Xiaoyin Shan, and Rachel Truitt. This work also required substantial use of the Penn Flow Cytometry Core Laboratory; in particular I thank Drew Bantly for assistance in fluorochrome panel design. I would like to thank my thesis committee including Dr. Daniel Hammer, Dr. John Gearhart, and Dr. Jason Burdick for insight, perspective, and scientific recommendations. Their depth of knowledge and commitment to rigorous science has helped improve and guide my work. Finally, I would like to thank my family. I thank my father for a rational mind, my mother for ambition and passion, and both for making the largest and longest-duration contribution to my life and thus this work. I thank my brother for our close friendship. I would also like to thank my fiancée and best friend Samra Zelman for happiness, compassion, respite, tolerance, and a little dog named Pig). iv ABSTRACT DYNAMICS OF ENDOGENOUS CARDIAC REPAIR AND METHODS FOR ENHANCED POST-INJURY CELL THERAPY Jeremy Alan Elser Kenneth Margulies, MD 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 apparentlyparadoxical studies and offers a unified estimate of cardiomyocyte turnover via a hybridmodel 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). v 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 cytometrybased engraftment assays enable future work in cardiac regeneration. vi TABLE OF CONTENTS DEDICATION... III ACKNOWLEDGMENT... IV ABSTRACT... V TABLE LEGEND... X FIGURE LEGEND... XI 1. OVERVIEW OF CARDIAC STEM CELL THERAPY Cardiac Injury Cardiomyogenesis in Physiological Homeostasis Cardiomyogenesis in Pathophysiologic Response Augmentation of the Repair Response Mobilization of Bone Marrow Progenitors Direct Delivery of Unfractionated Bone Marrow Cells Direct Delivery of Purified Stem Cells References SPECIFIC AIMS MODELING MYOCYTE TURNOVER IN THE ADULT HUMAN HEART Introduction Methods Modeling Approach Sources of Input Parameters Examination of Kajstura Manuscript in Isolation Using Hybrid Model Examination of the Bergmann Manuscript in Isolation Using Hybrid Model Turnover Models Simultaneously Compatible with Bergmann and Kajstura Data Hybrid Model Integration of Kajstura and Bergmann Models Results Hybrid Model Demonstrates Fidelity to Kajstura Model Parameter and Assumption Sensitivity in the Kajstura Model vii 3.3.3 New Considerations Applied to the Kajstura Model Hybrid Model Demonstrates Fidelity to Bergmann Dataset Parameter and Assumption Sensitivity in the Bergmann Model New Considerations Applied to the Bergmann Model Models Simultaneously Compatible with Bergmann and Kajstura Datasets Discussion References INJURY-DEPENDENT PROGENITOR CELL ENGRAFTMENT MEDIATED BY P- SELECTIN Introduction Methods Isolation of Mouse BMCs Isolated Perfused Mouse Heart for Ischemia Reperfusion Studies Flow Cytometry for IPMH Ischemia Reperfusion Studies Selectin Neutralization Studies in Isolated Perfused Hearts Histologic Analysis for IPMH Ischemia Reperfusion Studies Sorting of c-kit+ Cells from Mouse BMCs for Parallel Plate Flow Chamber Studies Parallel Plate Flow Chamber Studies Data Analysis for Parallel Plate Flow Chamber Studies Results Histological Evidence Infused Cell Engraftment Identification of Engrafted Cells in Non-ischemia Control Experiments Injury-Dependent Engraftment Selectivity for c-kit+/cd45+ Autologous BMCs Inhibition of P-selectin or L-selectin in Ischemic Conditions Rolling and Adhesion of c-kit+ Versus c-kit- BMCs on Selectins Discussion References LOCAL CHEMOKINE ENVIRONMENT MODIFICATION ENHANCES PROGENITOR CELL HOMING Introduction Rationale for Modification of the Injury-Site Chemokine Concentrations Hydrogel Delivery Systems for Extended Release of Chemokines: Stromal Derived Factor 1α and Progenitor Cell Chemotaxis: Summary of Work Materials and Methods Animals Macromer Synthesis Hydrogel Formation viii 5.2.4 BMC Isolation Experimental MyocardiaI Infarction (MI) In vivo BMC Homing Statistical Analysis Results: BMC Homing to the Remodeling Heart Discussion References ADJUVANT THERAPY FOR ARTIFICIAL ENHANCEMENT OF PROGENITOR CELL ENGRAFTMENT Introduction Methods Isolation of Mouse BMCs Isolated Perfused Mouse Heart (IPMH) for Ischemia Reperfusion Studies Flow Cytometry for Total BMC Retention Rate in Ischemia Reperfusion Studies Flow cytometry for Analysis of Biomarkers in IPMH Ischemia Reperfusion Studies Statistical Analysis Results Validation of BMC Recruitment Assay Peroxide Increases Engraftment of Bone Marrow Cells Viability of Heart Cells Post-Exposure to Peroxide Retention Selectivity Favors c-kit+ BMCs Discussion References SUMMARY, CONCLUSIONS, AND FUTURE WORK Summary Limitations Future Work APPENDIX - HYBRID MODEL JAVA CODE ix TABLE LEGEND Table 3.1. Modeled Patient Genders, Birth Years, and Lifespans Table 4.1 Selective Retention of c-kit+ Subsets at Various Reperfusion Durations.80 Table 6.1. Absolute Recovery Rates for Various Phenotypes x FIGURE LEGEND Figure 3.1. Input Atmospheric C14 Levels from Figure 3.2. Hybrid Model Automaton Algorithm Figure 3.3. The Hybrid Model Successfully Reproduces Kajstura Cell Dynamics Results from Kajstura Input Parameters...27 Figure 3.4. The Hybrid Model Successfully Reproduces Kajstura CM Age Distribution 28 Figure 3.5. Sensitivity of the Kajstura Analysis Estimate of CM Turnover to the Expansion Exponent Variable..29 Figure 3.6. Kajstura CM Count Trajectories and Sensitivity to Input Variables.. 30 Figure 3.7. Sensitivity of CM Age Histograms for Young, Middle-Aged, and Old Patients to Input Variables...32 Figure 3.8. Apoptosis-Dependent Definition of CM Turnover in the Kajstura Model Figure 3.9. Kajstura Turnover with Alternative Apoptosis Parameters...36 Figure 3.10 Kajstura Model Turnover Conclusions under Various Apoptotic CM Fractions.37 Figure Hybrid Model Validation Strategy Figure Hybrid Model Validation Results.40 Figure Cardiomyocyte Turnover Estimates using Bergmann Approach and Dataset with Bergmann Various Poly-ploidization Correction Factors...42 Figure Hybrid Model Simulations for the Bergmann Patient Dataset under Various Initial C14 Incorporation Assumptions...44 xi Figure Simulations Derived from Applying Pre-specified Annual CM Turnover Rates to Bergmann Model Analysis Method...47 Figure 3.16 Performance of Various Model Scenarios.. 53 Figure Impact of Assumptions and Parameter Measurement Uncertainty on Two Independent Models of CM Turnover...56 Figure Sensitivity of the Kajstura Analysis Estimate of CM Turnover to the Apoptosis Fraction Variable..57 Figure Sensitivity of the Kajstura Analysis Estimate of CM Turnover to the Expansion Exponent Variable..58 Figure 4.1. c-kit FMO on Filtered, RBC-Depleted Bone Marrow Cells...69 Figure 4.2. Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with PKH26GL+ BMCs...73 Figure 4.3. Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with PKH67GL+ BMCs and Co-staining for α-actinin and DAPI...74 Figure 4.4. Serial Gating to Identify Retained BMCs in Heart Digest..76 Figure 4.5. Biomarker Expression of Retained BMCs Following Sham-Ischemic and Ischemic Protocol 78 Figure 4.6. Cell Subtype Distribution in Bone Marrow, Sham-Ischemic Hearts, and Ischemic Hearts 79 Figure 4.7. Engraftment Ratios for Various BMC Subtypes at Reperfusion Durations of 0, 15, 30, 45, and 60 min...82 Figure 4.8. Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with PKH67GL+ BMCs and Co-staining for CD Figure 4.9. Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with PKH67GL+ BMCs and Co-staining for c-kit.85 xii Figure Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with unlabeled BMCs and Co-stained for c-kit and CD Figure Neutralizing Antibodies and Co-localization Implicate P-selectin in c- kit Selective Cell Engraftment.88 Figure Transverse Cryosection of Ischemia-Reperfused Mouse Heart (IPMH ) Infused with PKH67GL+ BMCs and Co-staining for P-selectin and c-kit..89 Figure Coexpression of PSGL-1 and c-kit in Bone Marrow Cells..91 Figure Rolling and Adhesion Dynamics of c-kit- and c-kit+ cells on P-Selectin and L-Selectin...93 Figure Rolling Flux and Rolling Concentration for c-kit- and c-kit+ BMCs over L-selectin and P-selectin Substrates..93 Figure 5.1. PKH- BMC Negative Controls for Flow Cytometry Gating Figure 5.2 In vivo model to quantify BMC homing Figure 5.3 Hydrogel Placement in situ Figure 5.4 Progenitor Cell Retention in vivo Figure 5.5. Size of PKH+ BMCs in the Blood Figure 5.6. Quantification of BMC Homing with Molecule Delivery to the Heart Figure 5.7. Visualization of PKH+ BMCs in the Myocardium 7 days after Systemic Infusion Figure 6.1. Flow Cytometry Gating Strategy for Heart Digest 123 Figure 6.2. Flow Cytometry Gating Strategy for Biomarker Evaluation..125 Figure 6.3. c-kit FMO on Filtered, RBC-Depleted Bone Marrow Cells..126 Figure 6.4. IPMH/Flow Cytometry Assay is Sensitive and Linear. 128 Figure 6.5. BMC Retention Rates are Enhanced by 3 mm Peroxide Exposure.129 xiii Figure 6.6. Native Heart Non-Viability is Unaffected by Peroxide Dosages Up to 3 mm Figure 6.7. Representative Biomarker Staining for BMC Perfusate and BMCs Retained in Heart for Various Protocols Figure 6.8. Cell Subtype Distribution in Bone Marrow, Sham-ischemic Hearts, Ischemic Hearts, and Sham-ischemic Hearts Receiving 3 mm Peroxide during the First 10 min of Reperfusion xiv CHAPTER 1 1. Overview of Cardiac Stem Cell Therapy 1.1 Cardiac Injury Heart failure (HF) is a leading cause of disability and death which accounts for approximately one million hospitalizations, over 50,000 deaths, and almost $35 billion in health care costs in the United States each year [1]. Despite therapeutic advances in the treatment of myocardial infarction, hypertension, valvular heart disease and cardiomyopathies, the prevalence of HF continues to grow. Mortality rates remain high with 50% of HF patients dying within five years of initial diagnosis [2]. Progressive loss of cardiomyocytes (CM), due to myocardial infarction (MI) and/or programmed cell death (apoptosis), is a feature of most causes of HF. Therefore, therapies to increase the number of functional CMs are expected to improve the course of this syndrome. The human heart was long believed to have virtually no capacity for new CM formation after childhood. This conclusion was drawn largely from the heart s inability to repair tissue damaged after myocardial infarction [3]. However, increasing evidence supports the existence of endogenous cardiac renewal and repair mechanisms that contribute to normal homeostasis and responses to pathological insults, which could be augmented to prevent heart failure. 1.2 Cardiomyogenesis in Physiological Homeostasis Overturning the dogma of the adult human heart as a terminally differentiated organ, Bergmann et al used a fate-mapping approach to demonstrate and quantify the rate 1 cardiomyocyte renewal [4]. Recognizing that an era of nuclear bomb tests between 1955 and 1963 (when the nuclear Test Ban Treaty was signed) produced a spike in atmospheric carbon14 (C14), these investigators used cardiac specific markers to identify and isolate CMs from hearts obtained at the time of autopsy. Because the C14 content of CM DNA should reflect the atmospheric the ambient C14 level at the time of cell formation, differences between the actual C14 content of DNA and the predicted level based on atmospheric C14 levels provided a means of identifying net cell replacement. In fact, higher than expected C14 CM DNA levels observed among those born before 1955 and the lower than expected C14 CM DNA levels observed those born after 1963 supported the replacement of some CMs during adult life. Even after accounting for previously described CM nuclear division (poly-ploidization), these investigators estimated that about 1% of CMs are replaced annually at the age of 25 and that this rate gradually decreases to about 0.45% annually by age 75. In 2010, Kajstura et al performed extensive histological analyses of 74 non-diseased hearts of various ages to evaluate the presence of a proposed class of cardiomyocyte stem cells (CSCs) and the fract
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