The development of the great vessels involves the retraction of the myocardial sheath and the migration, proliferation, and differentiation of cardiac neural crest (NC) and endocardial-derived mesenchymal cells. Evidence suggests that cell-cell and cell-extracellular matrix interactions between NC cells, cells of the outflow tract, and cardiac extracellular matrix are required for normal outflow tract development and may be mediated through specific growth factors. One of the growth factors is transforming growth factor-beta (TGF-beta). TGF-beta is usually secreted as a latent molecule that must be """"""""activated"""""""" in order to interact with receptors on target cells and this activation may be mediated by local proteolysis. Our data shows that in vitro, cardiac tissues secrete latent TGF-beta and NC cells can activate latent TGF-beta by pericellular proteolysis. We hypothesize that upon entering the outflow tract, nc cells activate latent cardiac TGF-beta by local proteolysis. The active TGF-beta then regulates myocardial cell proliferation and tenascin synthesis by the cardiac NC cells. To test this, we will demonstrate that (1) NC cell-proteolytic activity activates cardiac latent TGF-beta, (2) the temporal and spatial distribution of plasminogen activator and plasminogen activator-inhibitor expression is consistent with proteolytic activation of trophic factors during outflow tract development, (3) TGF-beta, basic fibroblast growth factor, and outflow tract conditioned medium regulate NC cell plasminogen activator activity, (4) TGF-beta, basic fibroblast growth factor, and NC cells mediate myocardial cell proliferation and survival and, (5) TGF-beta and outflow tract conditioned medium increases tenascin synthesis by NC cells. The proposed experiments will utilize fixed, sectioned avian embryos and primary cultures of outflow tract segments and NC cells in conjunction with TGF-beta bioassays, protease assays, and immunocytochemical and in situ hybridization methods to test these hypotheses. Results from these studies will enhance our understanding of the molecular and cellular mechanisms directing the development of great vessels and their associated valvular structures. This knowledge will be essential for developing improved prevention methods.
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