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    Investigating the Role of Camp-Signaling in the Regulation of Angiogenic Sprouting

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    Mackeil, Jodi
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    Abstract
    Angiogenesis, the growth of blood vessels from pre-existing vascular structures, can contribute adaptively or mal-adaptively to a myriad of conditions, including ischemic heart disease and cancer. During angiogenic sprouting, vascular endothelial cells (ECs) either function as “tip cells”, which guide blood vessel lumen formation, or proliferative “stalk cells” that lengthen the newly forming vessel. While the molecular mechanisms that govern tip and stalk cell specification are well-established, the systems that regulate individual EC functions during sprouting remain poorly understood. Importantly, the cAMP second messenger system has been implicated in the regulation of angiogenesis; however, the role of individual cAMP effector proteins, and the involvement of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms in controlling cAMP effector function during angiogenic sprouting, remain ill-defined. The studies presented in this thesis investigated the ability of the cAMP-signaling to regulate EC functions during angiogenic sprouting, Here, we identify a role for protein kinase A (PKA), but not exchange protein activated by cAMP (EPAC), as an intrinsic, negative regulator of EC sprouting both in vitro and ex vivo. At a cellular level, we demonstrate that constitutive PKA activity regulates EC sprouting by restricting podosome rosette biogenesis, proteolytic breakdown of the extracellular matrix (ECM), and the invasive capacity of vascular ECs. Upon investigating the molecular mechanisms through which PKA regulates EC sprouting function, we found that PKA reduces the level of active cdc42 by promoting its interaction with the regulatory inhibitor, Rho GDP-dissociation inhibitor α (RhoGDIα). Given that specificity of cAMP-signaling is achieved when PDEs act to locally control the “pool” of cAMP, which in turn activates a specific cAMP-effector protein, we investigated whether distinct PDEs were responsible for regulating PKA during angiogenic sprouting. We found that reduced expression of PDE3B or inhibition of PDE3 enzyme activity led to a corresponding increase in PKA activity, which markedly impaired EC sprouting. We further demonstrated that PDE3B and PKA interact within perinuclear regions of vascular ECs to regulate downstream cdc42 activity during EC sprouting. Overall, our work identifies a novel PDE3B-PKA signaling complex in vascular ECs and provides mechanistic insight into cAMP-dependent regulation of angiogenic sprouting.
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    http://hdl.handle.net/1974/26538
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    • Queen's Graduate Theses and Dissertations
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