Akt-mediated regulation of endothelial functions

 描述（由申请人提供）：血管稳态是一种涉及多种细胞类型和信号通路的基本生物学过程。与血管功能有关的许多因子（即剪切应力、细胞因子、生长因子刺激）通过PI 3 K/Akt（一种关键信号传导中间体）进行通信。内皮细胞（EC）主要表达Akt 1，这是参与心血管功能调节的主要亚型。我们以前的工作表明，Akt 1-/-;ApoE-/-小鼠动脉粥样硬化病变的扩展可能来自血管来源，这意味着疾病的进展是由EC或平滑肌细胞（SMC）谱系决定的。然而，SMC特异性Akt 1缺失不影响动脉粥样硬化病变的形成（未发表，合作者），指定内皮作为斑块发展的关键介质。因此，AIM 1将实施EC靶向Akt 1条件性小鼠模型，以证实内皮Akt 1表达在心血管疾病结局中的重要性。我们还报告了Akt 1-/-;ApoE-/-小鼠中eNOS磷酸化受损，与eNOS功能对心血管稳态的重要性相平行。虽然以前的遗传学研究表明eNOS作为Akt 1底物，但Akt 1介导的eNOS活化在动脉粥样硬化形成过程中的确切作用仍有待澄清。AIM 2将扩展这些遗传学研究，以直接测试Akt 1依赖的eNOS-S1176磷酸化对斑块形成的因果作用。使用表达“组成性活性”eNOS（S1176 D; Akt 1-/-; ApoE-/-）或“活性较低”eNOS（S1176 A; Akt 1-/-; ApoE-/-）的Akt 1基因敲除小鼠将描述EC衍生的NO产生对动脉粥样硬化病变发展的作用。最后，我们最近对内皮细胞裂解物的Akt磷酸化蛋白质组学分析表明，许多新的Akt底物对血管功能和/或渗透性具有潜在影响（例如VE-PTP，NUP 93）。AIM 3将整合我们的高通量磷酸化蛋白质组学阵列的结果，为以前未探索的Akt指导的内皮调节方法提供机制见解。了解Akt和新发现的靶蛋白之间的激酶-底物关系无疑将拓宽我们目前对Akt 1功能的认识以及对心血管功能的影响。总体而言，这 该提案旨在使用几种分子、细胞和遗传方法来破译内皮Akt 1表达/活性对EC生理学和心血管稳态的作用。培训内容：该提案概述了一个5年的职业发展计划，以促进候选人从指导博士后研究员过渡到独立的主要研究员。该候选人目前正在进行博士后培训的第四年，她继续参加定期研讨会并出席科学会议。这个应用程序建立在候选人的背景平滑肌生物学提供专门的重点内皮生理学和血管功能。本文提出的研究将在William C. Sessa博士(Yale他是内皮生物学和动脉粥样硬化形成领域公认的领导者。还建立了几个关键的合作，以提供与该提案有关的科学指导，包括卡洛斯费尔南德斯-埃尔南多博士，胆固醇和脂蛋白代谢专家，以及帕特里克拉斯克博士，核孔蛋白和核区室化专家。这项研究的结果不仅将促进我们对内皮PI 3 K/Akt信号传导的理解，还将提供宝贵的指导经验，为未来的独立研究人员奠定基础。

 DESCRIPTION (provided by applicant): Vascular homeostasis is an essential biological process involving multiple cell types and signaling pathways. Numerous factors implicated in vessel function (i.e. shear stress, cytokines, growth factor stimuli) communicate through PI3K/Akt, a key signaling intermediate. Endothelial cells (EC) express predominantly Akt1, the major isoform involved in regulation of cardiovascular function. Our previous work suggested that atherosclerotic lesion expansion in Akt1-/-;ApoE-/- mice was likely from vessel origin, implying disease progression is dictated by EC or smooth muscle cell (SMC) lineage. However, SMC-specific Akt1 deletion does not affect atherosclerotic lesion formation (unpublished, collaborator), designating the endothelium as a critical mediator of plaque development. AIM 1 will therefore implement EC-targeted Akt1 conditional mouse models to substantiate the importance of endothelial Akt1 expression in cardiovascular disease outcome. We also report impaired eNOS phosphorylation in Akt1-/-;ApoE-/- mice, paralleling the emerging importance of eNOS functionality for cardiovascular homeostasis. While previous genetic studies signify eNOS as an Akt1 substrate, the definitive role of Akt1-directed eNOS activation during atherogenesis remains to be clarified. AIM 2 will extend these genetic studies to directly test the causal role o Akt1-dependent eNOS-S1176 phosphorylation on plaque formation. Use of global Akt1-null mice expressing either the `constitutively-active' eNOS (S1176D; Akt1-/-; ApoE-/-) or `less-active' eNOS (S1176A; Akt1-/-; ApoE-/-) will delineate the role of EC-derived NO production on atherosclerotic lesion development. Lastly, our recent Akt phospho-proteomic analysis of endothelial lysates indicates a number of new Akt substrates with potential influence on vascular function and/or permeability (e.g. VE-PTP, NUP93). AIM 3 will integrate the results from our high-throughput, phospho-proteomic array to provide mechanistic insight on previously unexplored Akt-directed methods of endothelial regulation. Understanding the kinase-substrate relationship between Akt and the newly identified target proteins will undoubtedly broaden our current perspective of Akt1 function and the implications on cardiovascular function. Overall, this proposal aims to decipher the role of endothelial Akt1 expression/activity on both EC physiology and cardiovascular homeostasis using several molecular, cellular, and genetic approaches. Training: This proposal outlines a 5-year career development plan to facilitate the candidate's transition from a mentored postdoctoral fellow to an independent principle investigator. The candidate is currently in her 4th year of post- doctoral training where she continues to participate in regular seminars and present at scientific meetings. This application builds upon the candidate's background in smooth muscle biology by providing specialized focus on endothelial physiology and vascular function. The research proposed herein will be conducted under the continued mentorship of Dr. William C. Sessa, Ph.D. (Yale University, Department of Pharmacology), a recognized leader in the field of endothelial biology and atherogenesis. Several key collaborations have also been established for scientific guidance related to this proposal, including Dr. Carlos Fernandez-Hernando, Ph.D., an expert in cholesterol and lipoprotein metabolism, and Dr. Patrick Lusk, Ph.D., an expert on nucleoporins and nuclear compartmentalization. The results from this study will not only advance our understanding of endothelial PI3K/Akt signaling, but also provide an invaluable mentoring experience to create the foundations of a future independent researcher.