Rebuilding the human myocardium following pathological injury remains a significant challenge both at the level of basic research and clinical application. In the last decade, cell based therapy has emerged as a promising option for repairing damaged heart tissue, but a universally available treatment still eludes the medical profession. The work presented here investigates the cardiac response to injury in the context of crosstalk between stem cell and survival kinase signaling. The hypothesis of this thesis is that activated Notch participates in a HGF/PI3K/Akt mediated survival signaling response to cardiac injury, and that Notch signaling drives cardiac differentiation of cardiac progenitor cells (CPCs). Notch, a critical cue in the developing heart, is activated in damaged myocardium. Likewise, following myocardial infarction, c-Met, the tyrosine kinase receptor for hepatocyte growth factor (HGF), colocalizes with activated in nuclei of surviving cardiomyocytes. HGF stimulates Notch activity in neonatal rat cardiomyocytes (NRCMs) when applied in vitro and when injected into the intact heart. Moreover, exogenous Notch increases levels of cardiac phospho-Akt both in vitro and in vivo. Genetic tools were developed to manipulate activated Notch signaling and investigate regenerative/survival signaling crosstalk in intact heart as well as cardiac progenitor cells. A regulatable, intracellular Notch (KNIC) fused to the mutated estrogen receptor (mER) was created and tested for induction by tamoxifen. KNICmER was engineered into adenovirus for transient expression and lentivirus for stable integration into cardiac progenitor cells. Likewise, cardiac specific transgenic mice were created expressing KNICmER, and this construct will be subcloned downstream of the c-kit promoter to create a transgenic line with regulatable Notch activity in the progenitor population. These reagents will be applied in future studies aimed at improving the inherent reparative capacity of the mammalian heart as well as enhancing the regenerative success of adoptive transfer of cardiac progenitor cells into the damaged myocardium. Overall, this study demonstrates that activated Notch participates in cardiac protection following pathologic injury, in part by activation of the PI3K/Akt survival pathway, and that activated Notch also impacts cardiac progenitors by pushing them toward a cardiogenic lineage

Cardiovascular disease remains the primary cause of death in the United States and in most nations worldwide, despite ongoing intensive efforts to promote cardiac health and treat heart failure. Replacing damaged myocardium represents perhaps the most promising treatment strategy, but also the most challenging given that the adult mammalian heart is notoriously resistant to endogenous repair. Cardiac regeneration following pathologic challenge would require proliferation of surviving tissue, expansion and differentiation of resident progenitors, or transdifferentiation of exogenously applied progenitor cells into functioning myocardium. Adult cardiomyocyte proliferation has been the focus of investigation for decades, recently enjoying a renaissance of interest as a therapeutic strategy for reversing cardiomyocyte loss due in large part to ongoing controversies and frustrations with myocardial cell therapy outcomes. The promise of cardiac cell therapy originated with reports of resident adult cardiac stem cells that could be isolated, expanded and reintroduced into damaged myocardium, producing beneficial effects in preclinical animal models. Despite modest functional improvements, Phase I clinical trials using autologous cardiac derived cells have proven safe and effective, setting the stage for an ongoing multi-center Phase II trial combining autologous cardiac stem cell types to enhance beneficial effects. This overview will examine the history of these two approaches for promoting cardiac repair and attempt to provide context for current and future directions in cardiac regenerative research.

Stem cell mediated cardiac repair is an exciting and controversial area of cardiovascular research that holds the potential to produce novel, revolutionary therapies for the treatment of heart disease. Extensive investigation to define cell types contributing to cardiac formation, homeostasis and regeneration has produced several candidates, including adult cardiac c-Kit+ expressing stem and progenitor cells that have even been employed in a Phase I clinical trial demonstrating safety and feasibility of this therapeutic approach. However, the field of cardiac cell based therapy remains deeply divided due to strong disagreement among researchers and clinicians over which cell types, if any, are the best candidates for these applications. Research models that identify and define specific cardiac cells that effectively contribute to heart repair are urgently needed to resolve this debate. In this review, current c-Kit reporter models are discussed with respect to myocardial c-Kit cell biology and function, and future designs imagined to better represent endogenous myocardial c-Kit expression.

Cardiac fibroblast (CF) population heterogeneity and plasticity present a challenge for categorization of biological and functional properties. Distinct molecular markers and associated signaling pathways provide valuable insight for CF biology and interventional strategies to influence injury response and aging-associated remodeling. Receptor tyrosine kinase c-Kit mediates cell survival, proliferation, migration, and is activated by pathological injury. However, the biological significance of c-Kit within CF population has not been addressed. An inducible reporter mouse detects c-Kit promoter activation with Enhanced Green Fluorescent Protein (EGFP) expression in cardiac cells. Coincidence of EGFP and c-Kit with the DDR2 fibroblast marker was confirmed using flow cytometry and immunohistochemistry. Subsequently, CFs expressing DDR2 with or without c-Kit was isolated and characterized. A subset of DDR2⁺ CFs also express c-Kit with coincidence in ~ 8% of total cardiac interstitial cells (CICs). Aging is associated with decreased number of c-Kit expressing DDR2⁺ CFs, whereas pathological injury induces c-Kit and DDR2 as well as the frequency of coincident expression in CICs. scRNA-Seq profiling reveals the transcriptome of c-Kit expressing CFs as cells with transitional phenotype. Cultured cardiac DDR2⁺ fibroblasts that are c-Kit⁺ exhibit morphological and functional characteristics consistent with youthful phenotypes compared to c-Kit^- cells. Mechanistically, c-Kit expression correlates with signaling implicated in proliferation and cell migration, including phospho-ERK and pro-caspase 3. The phenotype of c-kit⁺ on DDR2⁺ CFs correlates with multiple characteristics of 'youthful' cells. To our knowledge, this represents the first evaluation of c-Kit biology within DDR2⁺ CF population and provides a fundamental basis for future studies to influence myocardial biology, response to pathological injury and physiological aging.

Enhancing cardiomyocyte survival is crucial to blunt deterioration of myocardial structure and function following pathological damage. PIM1 (Proviral Insertion site in Murine leukemia virus (PIM) kinase 1) is a cardioprotective serine threonine kinase that promotes cardiomyocyte survival and antagonizes senescence through multiple concurrent molecular signaling cascades. In hematopoietic stem cells, PIM1 interacts with the receptor tyrosine kinase c-Kit upstream of the ERK (Extracellular signal-Regulated Kinase) and Akt signaling pathways involved in cell proliferation and survival. The relationship between PIM1 and c-Kit activity has not been explored in the myocardial context. This study delineates the interaction between PIM1 and c-Kit leading to enhanced protection of cardiomyocytes from stress. Elevated c-Kit expression is induced in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1. Co-immunoprecipitation and proximity ligation assay reveal protein-protein interaction between PIM1 and c-Kit. Following treatment with Stem Cell Factor, PIM1-overexpressing cardiomyocytes display elevated ERK activity consistent with c-Kit receptor activation. Functionally, elevated c-Kit expression confers enhanced protection against oxidative stress in vitro. This study identifies the mechanistic relationship between PIM1 and c-Kit in cardiomyocytes, demonstrating another facet of cardioprotection regulated by PIM1 kinase.

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. The popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Despite widespread and increasing adoption of vaping among young adults there is little information on long term consequences of vaping and potential health risks. This study demonstrates Vaping-Induced Pulmonary Injury (VAPI) using commercial JUUL pens with flavored vape juice using an inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 weeks of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Transcriptional reprogramming includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress. Cardiac contractile performance for systemic output is moderately but significantly impaired, and the shows severe pulmonary side structural remodeling with chamber enlargement. This VAPI model with pulmonary circuit failure demonstrates mechanistic underpinnings of vaping-related pathologic injury.

An on-demand long-lived ultrasound contrast agent that can be activated with single pulse stimulated imaging (SPSI) has been developed using hard shell liquid perfluoropentane filled silica 500-nm nanoparticles for tumor ultrasound imaging. SPSI was tested on LnCAP prostate tumor models in mice; tumor localization was observed after intravenous (IV) injection of the contrast agent. Consistent with enhanced permeability and retention, the silica nanoparticles displayed an extended imaging lifetime of 3.3±1 days (mean±standard deviation). With added tumor specific folate functionalization, the useful lifetime was extended to 12 ± 2 days; in contrast to ligand-based tumor targeting, the effect of the ligands in this application is enhanced nanoparticle retention by the tumor. This paper demonstrates for the first time that IV injected functionalized silica contrast agents can be imaged with an in vivo lifetime ~500 times longer than current microbubble-based contrast agents. Such functionalized long-lived contrast agents may lead to new applications in tumor monitoring and therapy.

PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. The popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Despite widespread and increasing adoption of vaping among young adults, there is little information on long-term consequences of vaping and potential health risks. This study demonstrates vaping-induced pulmonary injury using commercial JUUL pens with flavored vape juice using an inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 wk of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Transcriptional reprogramming includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress. Cardiac systemic output is moderately but significantly impaired with pulmonary side ventricular chamber enlargement. This vaping-induced pulmonary injury model demonstrates mechanistic underpinnings of vaping-related pathologic injury.