The outcomes of cardiac repair with bone marrow mesenchymal stem cell (MSC), a highly heterogeneous cell population, have been variable and generally suboptimal. The efficacy of specific and homogenous MSC subsets defined by antigen expression has not been investigated. Based on preliminary data, our fundamental hypothesis states that transplantation of antigenically-defined CD45-/c-kit-/Sca- 1+/CD90+/CD105- (abbreviated as CD105-) MSC subpopulation will confer greater reparative and regenerative benefits after myocardial infarction (MI) by virtue of enhanced survival, increased retention, improved angiogenesis, greater differentiation into cardiac lineages, and favorable modulation of myocardial matrix. This hypothesis will be tested in a well-established cell culture model in vitro and a mouse model of reperfused MI in vivo, which will yield conclusive results.
Aim 1 will determine whether CD105- MSCs will be more resistant to apoptosis and washout. The susceptibility of murine bone marrow MSCs (Unfractionated, CD45-/c-kit-/Sca-1+/CD90+/CD105+ [abbreviated as CD105+, the precise antigenic control population], and CD105-) to apoptosis will be examined, and the underlying molecular basis elucidated. The expression of adhesion molecules on MSCs will be assessed, and cell retention in vivo will be tested following myocardial injection 2 d after a reperfused MI.
Aim 2 will investigate whether CD105- MSCs produce greater amounts of cardioprotective and angiogenic molecules, and acquire endothelial phenotype. Endothelial and cardiac commitment will be determined by morphology, and assessment of transcription factors and structural proteins. The molecular signaling underlying the angiogenic effects will be elucidated using specific inhibitors and siRNA, with particular attention to VEGFR2-activated pathways.
Aim 3 will establish whether transplantation of CD105- MSCs will induce superior cardiac repair in vivo, and identify the mechanistic basis in a definitive fashion. Unfractionated, and CD105+ and CD105- MSCs will be injected into the infarct borderzone 2 d after a reperfused MI in C57BL/6 mice. Serial echocardiography and a terminal hemodynamic study will be performed to assess left ventricular (LV) function and anatomy. LV structure, infarct size, fibrosis, and myocyte hypertrophy will be assessed by morphometry. Quantitative immunohistochemical methods will be used to precisely determine the contribution of myocyte salvage from apoptosis, angiogenesis, myocyte proliferation, myocyte regeneration, activation of cardiac progenitors, and modulation of calcium handling proteins. Focused proteomic analysis will be performed to identify novel changes in the matrix. For the first time, these studies will critically evaluate th biology of CD105- MSC subset and their efficacy in infarct repair in a comprehensive and thoroughly mechanistic fashion. The impact will be two-fold: (i) biologically, this project will yild novel insights into properties of antigenically-defined MSC populations;and (ii) clinically, the identification and validation of an optimal cell type for cardiac repair will benefit patients with ischemic heart disease and post-MI heart failure.
Ischemic heart disease is the single most prevalent cause of death and morbidity in all Western countries. Although recent studies indicate that therapy with adult bone marrow mesenchymal stem cells (MSCs) can repair dead heart muscle, the results have been variable, and the mechanisms remain unclear. If transplantation of antigenically-defined MSC subpopulation can induce superior cardiac repair after myocardial infarction, these cells may be utilized for heart repair in patients with ischemic heart disease and heart attacks. The results of the proposed studies will therefore be highly relevant for improving public health, and improving the length and quality of life of millions of patients with ischemic heart disease and heart failure.
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