Abnormal development of the cardiac mesenchyme (CM) has been implicated as a primary cause of heart malformations which account for the majority of clinically significant birth defects. The CM of the AV canal is derived from endothelium and forms the valuve leaflets and internal partitions of the heart. The formation of CM derives from a tissue interaction between the endothelium and the myocardium which is """"""""driven"""""""" by inductively active components of the myocardial basement membrane (MBM). Recently we have succeeded in extracting a factor from the MBM which induces cultured AV endothelium to transform into prevalvular mesenchyme following the same cascading sequence of cellular events as occur in situ. We have also identified the factor in conditioned media of myocardial cultures. Based on these and other data compiled during the tenure of this grant we propose to: (1) biochemically and immunologically characterize fractionated extracts of the MBM and CM to determine if their biological activity resides in a specific group of proteins that may be complexed into a 30 nm multicomponent particle which remarkably is also seen in situ; (2) determine if a unique isoelectric form of fibronectin functions in the assembly of the inductively active, MBM proteins into a multicomponent particle or serves as a carrier for lower molecular weight proteins that induce endothelial transformation into mesenchyme; (3) identify the precursors of putative subsets of the endothelium whose differentiatial response to MBM extracts may be programmed by extracellular basement membrane proteins produced by the foregut endoderm; In regard to this hypothesis, we have made an novel observation that normally nontransforming ventricular endothelium can be induced to form mesenchyme by co-culture with endoderm and myocardium and (4) determine if the high (greater than 90%) frequency of valvular and septal malformations which occur in a trisomiy 16 mouse model of Down's syndrome result from altered CM cellular dynamics and/or MBM components. These hypotheses will be tested in vitro using a reproducible endothelial activation assay developed in our lab and in vivo by localization of specific markers (e.g. monoclonal antibodies) to endothelial cells, MBM proteins or endodermal basement membrane components or by alterations of in situ relationships by microinjection and microsurgical techniques. The experiments in this proposal are designed to explain how macromolecules formed during early embryogenesis could mechanistically regulate the development and differentiation of endothelial precursor cells into cardiac mesenchyme and prevalvular fibroblasts.
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