The long-term goal of this project is to understand the role that gap junction-mediated communication plays in regulating vascular responses and to define the contributions of specific gap junction proteins. Gap junctions are clusters of intercellular channels, comprised of connexin (Cx) proteins, that connect the cytoplasm of adjacent cells, allowing the direct cell-to-cell transfer of small molecules. Alterations in Cx expression and function have been correlated with hypertension, ischemia, inflammation, atherosclerosis, atrial fibrillation, and heart failure. In addition, Cx mutations or altered Cx expression caused by other gene mutations could be responsible for congenital defects of the heart and vasculature. This project takes advantage of the availability of knockout mice lacking specific vascular Cxs to study Cx function in the vasculature. In the first specific aim, the role of Cx37 and Cx40 in vascular development and angiogenesis is investigated. A variety of methods are used to explore the mechanism of abnormal vessel formation in mice lacking both Cx37 and Cx40, including use of an endothelial cell-specific reporter gene, cell proliferation assays, and apoptosis assays. Primary endothelial cell cultures are prepared to directly compare proliferation and apoptosis in the presence or absence of specific Cxs. The effect of exogenous expression of Cxs on cell growth and apoptosis is also examined in cell cultures. Angiogenic responses in Cx-deficient and wild-type mice are compared using both an in vivo assay and an explant approach.
A second aim i s to determine if Cx37 and Cx40 form channels of mixed composition and unique functional properties in vascular endothelial cells, using electrophysiological and biochemical methods. Heteromeric channels could play an important role in determining the types of signals that are communicated during vascular development, angiogenic responses, and vasomotor responses. Understanding the phenotypes of Cx deficient mice may provide insight into congenital vascular defects in humans and could also shed light on the processes that regulate normal vascular development. A better understanding of vascular Cxs could lead to new insights, treatments, and prevention for congenital vascular defects and cardiovascular disease. Finally, these studies could also lead to new approaches to control vasculogenesis and angiogenesis in other clinically important settings, such as anti-angiogenesis treatments designed to fight cancer.
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