Akt-mediated regulation of endothelial functions

Akt 介导的内皮功能调节

• 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
• 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

 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（由适用提供）：血管稳态是涉及多种细胞类型和信号通路的必不可少的生物学过程。血管功能（即剪切应力，细胞因子，生长因子刺激）实施的许多因素通过PI3K/AKT（一种关键信号中间体）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同工型。我们以前的工作表明，Akt1 - / - ; ApoE-/ - 小鼠中的动脉粥样硬化病变的扩展可能来自血管起源，这表明疾病进展取决于EC或平滑肌细胞（SMC）谱系。但是，SMC特异性的AKT1缺失不会影响动脉粥样硬化病变的形成（未发表，合作者），将内藻设计为斑块发育的关键介体。因此，AIM 1将实施靶向EC的AKT1条件小鼠模型，以证实内皮AKT1表达在心血管疾病结果中的重要性。我们还报告了Akt1 - / - ; apoE-/ - 小鼠中的ENOS磷酸化受损，这与ENOS功能对心血管稳态的新兴重要性并行。虽然先前的遗传研究表示eNOS是AKT1底物，但在动脉粥样硬化期间，Akt1定向的eNOS激活的明确作用仍然待阐明。 AIM 2将扩展这些遗传研究，以直接测试斑块形成上Akt1依赖性eNOS-S1176磷酸化的因果作用。使用表达“宪法活性” eNOS（S1176D; akt1 - / - ; apoe - / - ）或“较小活动” eNOS（S1176A; akt1; akt1 - / - ; apoE-; apoe---）的全局AKT1-null小鼠的使用将无法描述Ec衍生作用在Athersocter of Athersot of the siion lesion lession的作用。最后，我们最近对内皮裂解物的AKT磷酸蛋白质分析表明，许多新的Akt底物对血管功能和/或渗透性有潜在影响（例如VE-PTP，NUP93）。 AIM 3将整合我们的高通量磷酸化阵列阵列中的结果，以提供有关先前出乎意料的内皮调节方法的机械见解。了解AKT与新鉴定的靶蛋白之间的激酶 - 基底关系无疑将扩大我们当前对AKT1功能的观点以及对心血管功能的影响。总体而言，这 建议旨在使用几种分子，细胞和遗传方法来解读内皮AKT1表达/活性在EC生理和心血管稳态上的作用。培训：该提案概述了一项为期5年的职业发展计划，旨在促进候选人从受过指导的博士后研究员到独立的主要研究员的过渡。该候选人目前正在她的博士后培训的第四年，她继续参​​加常规的半手，并参加了科学会议。该应用程序通过专门关注内皮生理学和血管功能的专门关注，以平滑肌生物学的背景为基础。本文提出的研究将以威廉·C·塞萨（William C. Sessa）博士的持续心态进行。 （耶鲁大学，药理学系），内皮生物学和动脉粥样硬化领域的公认领导者。还建立了与该提案有关的科学指导的几项关键合作，包括胆固醇和脂蛋白代谢的专家Carlos Fernandez-Hernando博士，以及核孔林和核核心和核分类的专家Patrick Lusk博士。这项研究的结果不仅会提高我们对内皮PI3K/AKT信号传导的理解，而且还提供了宝贵的心理体验来创造未来独立研究人员的基础。