Akt-mediated regulation of endothelial functions

Akt 介导的内皮功能调节

基本信息

批准号：
9916034
负责人：
Monica Y Lee
金额：
$ 24.9万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9916034
关键词：
1-Phosphatidylinositol 3-Kinase Ablation Adult Apolipoprotein E Arterial Fatty Streak Biochemical Biological Biological Process Biology Blood Vessels CRISPR/Cas technology Cardiovascular Diseases Cardiovascular Pathology Cardiovascular Physiology Cardiovascular system Cell Lineage Cell physiology Cells Cellular biology Cholesterol Collaborations Consequentialism Cre-LoxP Data Development Development Plans Disease Outcome Disease Progression Doctor of Philosophy Endothelial Cells Endothelium Excision Foundations Functional disorder Future Generations Genetic Genetic study Growth Factor Homeostasis Impairment In Vitro Inflammation Investigation Knockout Mice Lesion Lipoproteins Mammalian Cell Measures Mediating Mediator of activation protein Mentors Mentorship Metabolism Methods Molecular Mus Mutant Strains Mice Mutation Myography Nitric Oxide Nuclear Nuclear Pore Nuclear Pore Complex Proteins Pathogenesis Pathologic Processes Pattern Permeability Pharmacology Phenotype Phosphorylation Phosphotransferases Physiology Play Postdoctoral Fellow Production Protein Isoforms Proteins Receptor Protein-Tyrosine Kinases Regulation Reporting Research Research Personnel Retinal Role Signal Pathway Signal Transduction Site Smooth Muscle Smooth Muscle Myocytes Stimulus Techniques Testing Therapeutic Training Universities Vascular Permeabilities Work atherogenesis atheroprotective cardiovascular health career development cell behavior cell growth cell type cytokine endothelial dysfunction experience gain of function genetic approach genetic manipulation in vivo insight meetings migration mouse model novel phosphoproteomics pleiotropism post-doctoral training selective expression shear stress vascular endothelial protein tyrosine phosphatase virtual

项目摘要

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.

描述（由申请人提供）：血管稳态是一个重要的生物过程，涉及多种细胞类型和信号通路，涉及血管功能的许多因素（即剪切应力、细胞因子、生长因子刺激）通过关键信号传导中间体 PI3K/Akt 进行通信。内皮细胞 (EC) 主要表达 Akt1，这是参与心血管功能调节的主要异构体。 Akt1-/-;ApoE-/- 小鼠可能来自血管，这意味着疾病进展由 EC 或平滑肌细胞 (SMC) 谱系决定。然而，SMC 特异性 Akt1 缺失不会影响动脉粥样硬化病变的形成（未发表，合作者）。因此，将内皮细胞设计为斑块形成的关键介质，因此将实施 EC 靶向 Akt1 条件小鼠模型，以证实内皮 Akt1 的重要性。我们还报告了 Akt1-/-;ApoE-/- 小鼠中 eNOS 磷酸化受损，这与 eNOS 功能对心血管稳态的重要性日益凸显，而之前的遗传学研究表明 eNOS 作为 Akt1 底物，是 Akt1 的决定性作用。 Akt1 介导的 eNOS 在动脉粥样硬化形成过程中的激活仍有待阐明，AIM 2 将扩展这些基因研究，以直接测试 Akt1 依赖性的因果作用。 eNOS-S1176 磷酸化对斑块形成的影响使用表达“组成型活性”eNOS（S1176D；Akt1-/-；ApoE-/-）或“低活性”eNOS（S1176A；Akt1-/）的整体 Akt1 缺失小鼠。 -; ApoE-/-) 将描述 EC 衍生的 NO 产生对动脉粥样硬化病变的作用最后，我们最近对内皮裂解物进行的 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/或通透性具有潜在影响（例如 VE-PTP、NUP93），将整合我们的高通量结果。吞吐量，磷酸化蛋白质组阵列，为以前未探索的 Akt 导向的内皮调节方法提供机制见解，了解激酶与底物之间的关系。 Akt 和新发现的靶蛋白无疑将拓宽我们目前对 Akt1 功能及其对心血管功能影响的认识。该提案旨在使用多种分子、细胞和遗传方法破译内皮 Akt1 表达/活性对 EC 生理学和心血管稳态的作用。培训：该提案概述了一个 5 年职业发展计划，以促进候选人从受指导的博士后过渡。该候选人目前正处于博士后培训的第四年，她将继续参加定期研讨会并出席科学会议。该申请以候选人的背景为基础。通过专门关注内皮生理学和血管功能，本文提出的研究将在 William C. Sessa 博士（耶鲁大学药理学系）的持续指导下进行，他是该领域公认的领导者。内皮生物学和动脉粥样硬化领域还建立了几项关键合作，以提供与该提案相关的科学指导，其中包括胆固醇和脂蛋白代谢专家 Carlos Fernandez-Hernando 博士，以及Patrick Lusk 博士是核孔蛋白和核区室化方面的专家。这项研究的结果不仅将增进我们对内皮 PI3K/Akt 信号传导的理解，而且还提供宝贵的指导经验，为未来奠定基础。独立研究员。