PI’s: Michael Davis & Mary Wagner
Cardiomyopathy, long since believed to be a problem in adults following injury or aging, has recently moved to the forefront of the pediatric population. Improved repair of cardiac birth defects, as well as enhanced treatment of acute cardiac injury in the pediatric population has led to increases in the incidence of cardiomyopathy in the young. Cardiomyopathy is the leading reason for transplantation in the young, and the mortality and transplant rate after 2-years is 40%.1 In addition to cardiac effects, ventricular dysfunction can also lead to end-organ damage through reduced blood flow.2, 3 As the damage is quite localized, therapeutics that can deliver local factors are needed. Cell based therapies have been met with enthusiasm, however much debate still lingers on the optimal delivery method of cells and exact cell type which holds the most promise. Indeed, many cells most likely diffuse away from the site of injection, making biomaterial-based grafts more necessary.4 These grafts, while promising, have many shortcomings when combined with cell therapy including poor cell engraftment, survival and differentiation.
In vivo, stem and progenitor cells exist in local niches, be it bone marrow or tissue.5, 6 These niches likely provide specific cues that influence cell function. Cardiac progenitor cells (CPCs), a progenitor cell found in the myocardium that may contribute to healing following infarction, are one such cell type. These cells have cardiogenic gene expression, as well as the stem cell marker c-kit. Following injury, these cells migrate to the area of damage and attempt to replace the lost tissue.6, 7 While the endogenous response seems inadequate, recent clinical trials have begun to determine whether delivery of these cells repairs and/or regenerates damage myocardium in adults.8 Here we propose to use a novel injectable hydrogel that contains immobilized Notch ligands. Thus, the central hypothesis of this proposal is that human CPCs from pediatric patients delivered within a hydrogel containing Notch signals will enhance survival, differentiation, and regenerative ability, leading to enhanced cardiac function. This hypothesis is based on our preliminary data showing immobilized Notch ligand in a self- assembling hydrogel activates Notch downstream targets and enhances both cardiac and vascular gene expression. Interestingly, the differentiation of these cells can be tuned with the mechanical properties of the hydrogel. These hydrogels have been shown to be non-toxic from mice all the way to pigs and the Davis laboratory has several publications involving delivery of self-assembling peptides following myocardial injury.9-14 The overall hypothesis of this seed grant will be tested as follows:
Specific Aim 1: Characterization of a bioactive hydrogel with immobilized Jagged ligands for activation of Notch in human CPCs in 3D. We have identified a 30 amino acid region of Jagged (JAG) that is capable of activating Notch signaling in rat CPCs. We have received IRB approval to isolate human CPCs from pediatric patients. Cells will be embedded in hydrogels containing the JAG or scrambled peptide (0.1, 1, 5, and 10% w/w) and Notch activation will be determined by cleavage of the Notch intracellular domain, and expression of Notch effectors (Hes and Hey family). To determine the effects of immobilized JAG on CPCs, cells will be cultured in JAG (or scrambled peptide) containing hydrogels and gene expression of major cardiac, endothelial, and smooth muscle cells will be measured. Functional measurements will be taken (contractility, Ca++) as well as determination of cell survival/death.
Specific Aim 2: Human CPCs transplanted within hydrogels will persist longer, demonstrate enhanced differentiation, and improve cardiac function in vivo. To determine functional efficacy of JAG hydrogels in vivo, adolescent male nude rats (150g) will be subjected to pulmonary artery banding to induce right ventricular heart failure. After onset of dysfunction, animals will be randomized in to several treatment groups including saline, JAG and scrambled hydrogels alone, and human CPCs in either hydrogel. Cells will be infected with a lentivirus encoding luciferase for quantitative cell tracking using in vivo imaging. Function will be measured at 7, 14, and 28 days post-implantation using echocardiography and end-point invasive hemodynamic measurements will be performed.
Currently, CPC therapy is in early clinical trials in adults. However; the increased incidence of cardiomyopathy in the pediatric population and the need for therapies that improve function present a unique opportunity for CPC therapy. Improving cardiac function in these patients may improve quality of life, allow for longer time to find suitable donors, or potentially prevent/delay right ventricular failure following corrective surgery. With the GMP cell facility at Emory, this seed grant has the potential to translate rapidly to the clinic.