Our previous studies provide direct evidence that gap junction communication between endothelial cells and the mesenchymal cells that they recruit, via Cx43-containing channels, mediates the activation of TGF-beta, which is required for endothelial-induced mural cell differentiation during blood vessel assembly. In the proposed studies, we focus on elucidating the mechanism(s) by which gap junction communication regulates these processes. To gain insight into the mechanism(s), we will determine whether other Cx proteins expressed in the vasculature (Cx45, Cx40, Cx37), which exhibit distinct selectivity and regulatory properties, are capable of supporting these processes. Our Preliminary data, to date, demonstrate that Cx40 does not. Thus, our overall hypothesis, based on Preliminary data and the phenotypes of mice lacking specific Cx proteins, is that only Cx43- and Cx45-containing gap junction channels between endothelial and mesenchymal cells can mediate TGF-beta activation and endothelial-induced mural cell differentiation. We further hypothesize that TGF-beta activation is mediated either via the exchange of cytoplasmic signals through Cx43- and Cx45- containing channels that are excluded by other channel types (Cx40 and Cx37), or via signaling events involving the regulation of the unique cytoplasmic domains of Cx43 and Cx45 proteins. To address these issues, we will measure differences in the selectivity of gap junction channels that mediate TGF-beta activation and endothelial-induced mural cell differentiation (Cx43-containing) vs. those that do not (Cx40-containing). We will examine the role of the cytoplasmic, regulatory region of Cx43 in the activation of TGF-beta via testing the ability of Cx43-/- mesenchymal cell transfectants that express Cx43 lacking the entire cytoplasmic, regulatory region or containing specific point mutations within that region, to form functional gap junction channels with endothelial cells, and mediate TGF-beta activation and endothelial-induced mural cell differentiation. Finally, using the information obtained in the aforementioned studies we will examine the signaling events supported by gap junctions that lead to TGF-b activation. Our studies will reveal the mechanism(s) by which gap junction communication regulates blood vessel assembly. Insights gained from these studies will be applicable to ex vivo vascular tissue engineering and will aid in the development of strategies to promote or suppress vessel formation for the treatment of prevalent pathologies.
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