Coronary artery disease is the leading cause of death in persons over 65. Myocyte loss and endothelial dysfunction contributes to the increased severity of cardiovascular disease in the aging population. Approaches specifically aimed at replacing the necrotic myocardium with adult derived bone marrow stem (BMS) cells for transdifferentiation into cell lineages of the myocardium could provide a basis for effective treatment of age-related diseases. The major hypotheses to be tested are: 1) BMS cells become scarce in aging animals but have the potential to acquire cardiac phenotypes; 2) BMS cells transdifferentiate in old myocardium and improve cardiac function; 3) Mobilization of BMS cells in aging myocardium promotes neoangiogenesis and myocyte repair; 4) Mild cellular stress stimulates growth factors resulting in neoangiogenesis as well as proliferation of BMS cells into cardiac phenotypes. We will utilize young adult and old mice to characterize age related changes in BMS cells and their potential to grow in the old myocardium into cardiac phenotypes. Where feasible, BMS cells from transgenic mice expressing enhanced green fluorescent protein (GFP) will be used. The origin of cells in the regenerating myocardium following BMS cell transplantation will be determined by the expression of GFP. The coculture cell model will be used to study the biology of BMS cells and their differentiation potential into cardiac phenotypes after exposure to different cardiac microenvironment. Both isolated perfused myocardium and left anterior descending coronary artery ligation model will be used to determine the effects of BMS cell engraftment and differentiation in cardiac function and pathology. The regenerating myocardium will be determined by GFP, BrdU and Ki67 immunocytochemistry. Both latter markers label cells in S phase indicative of cell proliferation. Myocyte proteins, nestin and desmin will be localized. The maturing myocytes will be determined by expression of myocyte enhancer factor 2, transcription factor (GATA-4) and early marker of myocyte development, CSX/NKx2.5 ; vascular endothelial cells involved in angiogenesis will be recognized by factor VIII, cadherin and CD34 antibodies ; and smooth muscle will be identified by anti alpha smooth muscle actin. The coupling between myocytes will be assessed with connexin 43 antibodies and carboxyfluorescein dye. The proposed studies will provide a comprehensive information about the biology of BMS cells in the aging myocardium and their potential to transdifferentiate into cell lineages of the host organ for stem cell-based strategies in the treatment of aging-related diseases.
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