Akt-mediated regulation of endothelial functions

Akt 介导的内皮功能调节

• 批准号: 9014192
• 负责人: Monica Y Lee
• 金额: $ 10.49万
• 依托单位: YALE UNIVERSITY
• 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9014192
• 关键词: 1-Phosphatidylinositol 3-Kinase; Ablation; Adult; Affect; Apolipoprotein E; Arterial Fatty Streak; Biochemical; Biological; Biological Process; Biology; Blood Vessels; Breeding; CRISPR/Cas technology; Cardiovascular Diseases; Cardiovascular Pathology; Cardiovascular Physiology; Cardiovascular system; Cell Lineage; Cell physiology; Cells; Cellular biology; Collaborations; 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; Homeostasis; In Vitro; Inflammation; Investigation; Knockout Mice; Lesion; 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; Technology; Testing; Therapeutic; Training; Universities; Vascular Permeabilities; Work; atherogenesis; cardiovascular health; career development; cell behavior; cell growth; cell type; cytokine; endothelial dysfunction; experience; gain of function; genetic approach; genetic manipulation; in vivo; insight; lipoprotein cholesterol; meetings; migration; mouse model; novel; phosphoproteomics; pleiotropism; post-doctoral training; selective expression; shear stress; vascular endothelial protein tyrosine phosphatase

 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(申请人提供)：血管动态平衡是涉及多种细胞类型和信号通路的基本生物学过程。许多与血管功能有关的因子(如剪切力、细胞因子、生长因子刺激)通过PI3K/Akt这一关键的信号中间产物进行交流。内皮细胞(EC)主要表达Akt1，Akt1是参与心血管功能调节的主要亚型。我们以前的工作表明，Akt1-/-；ApoE-/-小鼠动脉粥样硬化病变的扩大可能来自血管起源，这意味着疾病的进展是由EC或SMC谱系决定的。然而，SMC特异的Akt1缺失并不影响动脉粥样硬化病变的形成(未发表，合作者)，这表明内皮细胞是斑块发展的关键介质。因此，AIM 1将实施EC靶向Akt1条件性小鼠模型，以证实内皮Akt1表达在心血管疾病预后中的重要性。我们还报告了Akt1/-；ApoE-/-小鼠eNOS磷酸化受损，与eNOS功能对心血管内稳态的新出现的重要性平行。虽然以前的遗传学研究表明eNOS是Akt1的底物，但Akt1诱导的eNOS激活在动脉粥样硬化形成过程中的确切作用仍有待阐明。目的2将扩展这些遗传学研究，以直接测试Akt1依赖的eNOS-S1176磷酸化在斑块形成中的因果作用。使用整体Akt1缺失的小鼠表达eNOS(S1176D；Akt1-/-；ApoE-/-)或不太活跃的eNOS(S1176A；Akt1-/-；ApoE-/-)，将描述EC来源的NO产生在动脉粥样硬化病变发展中的作用。最后，我们最近对内皮细胞裂解产物进行的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/或通透性有潜在影响(例如VE-PTP，NUP93)。Aim 3将整合我们的高通量、磷酸蛋白质组阵列的结果，以提供对以前未探索的Akt指导的内皮调节方法的机械性见解。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Akt1功能及其对心血管功能的影响的视角。总体而言，这 该提案旨在利用几种分子、细胞和遗传学方法破译内皮Akt1表达/活性在EC生理和心血管内稳态中的作用。培训：本提案概述了一个5年的职业发展计划，以促进候选人从一名有指导的博士后研究员转变为一名独立的原则研究员。这位候选人目前正处于博士后培训的第四个年头，在那里她继续定期参加研讨会和科学会议。这一应用程序建立在候选人的平滑肌生物学背景之上，提供内皮生理学和血管功能的专门关注。在此提出的研究将在威廉·C·塞萨博士(耶鲁大学药理学系)的持续指导下进行，他是内皮生物学和动脉粥样硬化领域公认的领先者。与这一提议相关的科学指导方面也建立了几个关键的合作，包括胆固醇和脂蛋白代谢专家卡洛斯·费尔南德斯-埃尔南多博士和核孔素和核区划专家帕特里克·卢斯克博士。这项研究的结果不仅将促进我们对内皮PI3K/Akt信号转导的理解，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。