The primary goal of this proposal is to equip the candidate with the tools and language of molecular biology and cellular imaging directed towards the study of the molecular mechanisms of atherogenesis. This training will be done in the context of the proposed project focusing on the role of the protein kinase Akt in endothelial cell activation. The candidate is a clinically trained cardiologist with a research background in physical organic chemistry and laser spectroscopy. Her broader research interest lies in applying molecular biology and fluorescence microscopy and spectroscopy to examine protein-protein interactions critical in the progression of cardiovascular disease. Educational objectives of this proposal include developing the candidate's proficiency in the techniques of state of the art molecular biology and confocal fluorescence microscopy and expanding her knowledge base in vascular biology and signal transduction to lay the foundation for a career in cardiovascular research. Endothelial cell (EC) activation is thought to play an important role in atherogenesis through enhanced vascular leukocyte recruitment. Recent studies suggest Akt can both inhibit and enhance inflammation through activation of eNOS and NF-KB, respectively. The net effect of Akt activation may critically depend on the underlying endothelial context and published studies to date have been performed only in normal, not dysfunctional EC or vessels. The scientific goal of the current proposal is to define the effects of Akt activation on EC activation and monocyte recruitment in models of endothelialdys function. This proposal is based on three hypotheses: 1)Akt plays an important role in the intracellular signaling pathways mediating endothelial activation. 2)Endothelial dysfunction shifts the balance of Akt activation towards a pro-inflammatory phenotype. 3)This balance is a critical determinant of monocyte recruitment and the progression of atherosclerosis. To test these hypotheses, EC expression of mutant and wild-type Akt constructs will be achieved using recombinant adenoviral gene transfer in models of monocyte recruitment and atherogenesis. In vitro and in vivo studies will utilize LDLR and eNOS knockout models of endothelial dysfunction. As Akt activation has both salutary and detrimental effects on endothelial cell activation, a greater understanding of the mechanisms by which Akt governs endothelial activation and the factors that determine this balance, may provide insight into novel therapeutic approaches to atherosclerosis. In summary, the goals of this proposal are to prepare the candidate for the transition to independent investigator by providing her with the support and time to further the breadth and depth of her knowledge in vascular biology and acquire the technical skills of molecular biology and cellular imaging to complement her background in fundamental chemistry as she embarks on a career at the interface of biological chemistry and cardiology.
