An isoform-specific, conditional deletion approach to decipher Akt1 and Akt2 func

破译 Akt1 和 Akt2 功能的异构体特异性条件删除方法

• 批准号: 8526932
• 负责人: Monica Y Lee
• 金额: $ 4.92万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526932
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acute; Address; Adult; Affect; Amino Acids; Angiogenesis Inhibitors; Arteries; Biological Process; Blood Vessels; Breeding; Cardiovascular Diseases; Cell Lineage; Cells; Complement; Development; Embryo; Embryonic Development; Endothelial Cells; Endothelium; Excision; Exhibits; Financial compensation; Future; Generations; Genes; Genetic; Growth; Growth Factor; Growth and Development function; Human Pathology; Impairment; Insulin Resistance; Ischemia; Knock-out; Knockout Mice; Limb structure; Mammalian Cell; Measures; Mediating; Metabolism; Modeling; Molecular; Mus; Myography; Nature; Nitric Oxide; Pathologic Neovascularization; Pattern; Perinatal; Phenotype; Phosphorylation; Physiological; Process; Production; Protein Isoforms; Receptor Protein-Tyrosine Kinases; Regulation; Research; Resistance; Retinal; Role; Signal Pathway; Signal Transduction; Smooth Muscle Myocytes; Stimulus; Structure; Techniques; Testing; Therapeutic; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular remodeling; Vascularization; Work; angiogenesis; cadherin 5; cancer therapy; cell growth; cell type; cytokine; design; diabetic; human MYH11 protein; in vivo; migration; mouse model; postnatal; public health relevance; response; retinal angiogenesis; shear stress; tool

DESCRIPTION (provided by applicant): Vascularization is an essential process involved in embryonic development and vascular remodeling. Numerous stimuli (i.e. shear stress, cytokines, growth factors) promote vessel growth through PI3K-dependent Akt activation. Activated Akt (Akt1/Akt2/Akt3) targets a wide range of substrates involved in key endothelial functions, including regulation of vascular tone, angiogenesis, and cellular recruitment to vessel walls. Endothelial cells (EC) and vascular smooth muscle cells (SMC) express predominantly the Akt1 isoform. Several groups have demonstrated impaired migratory and proliferative responses in cultured Akt1-null EC and SMC, thereby supporting the pro-angiogenic role of PI3K/Akt signaling across vascular cell types. PI3K/Akt signaling is also critical during adult angiogenesis, as global loss of Akt1 results in impaired ischemia- and VEGF-induced angiogenesis. Global knockout models of the Akt isoforms have helped unravel the physiological roles of Akt. However, the emergence of nonredundant phenotypes along with ubiquitous expression of Akt1 limits our understanding of Akt1 function in a cell-specific manner. We have therefore generated conditional Akt1flox/flox mice to examine the importance of EC- and SMC-specific Akt1 loss during embryonic and postnatal angiogenesis. We have additionally bred the conditional Akt1 mice to an Akt2-null background, effectively creating an inducible double knockout mouse to address possible compensation effects. We intend to examine how EC-specific Akt1 deletion affects the retinal vasculature and adult ischemia-induced angiogenesis. Given that the endothelium regulates vascular tone through Akt-dependent nitric oxide production, we will include vascular reactivity studies to measure consequent effects on vessel tone. The hind-limb ischemia model will also be applied to investigate how the SMC-specific loss of Akt1 influences resulting arteriogenesis/angiogenesis and vascular remodeling. These studies will be further complemented with use of our inducible double knockout mice to examine the consequent effect of dual Akt1/2 loss. Thus, the overall focus of this proposal is to elucidate the roles of Akt1 and Akt2 in two major vascular cell types (EC and SMC) using several established molecular, cellular, and genetic approaches.

描述（由申请人提供）：血管化是涉及胚胎发育和血管重塑的重要过程。许多刺激（即剪切应力、细胞因子、生长因子）通过 PI3K 依赖性 Akt 激活促进血管生长。激活的 Akt (Akt1/Akt2/Akt3) 靶向涉及关键内皮功能的多种底物，包括调节血管张力、血管生成和细胞募集至血管壁。内皮细胞 (EC) 和血管平滑肌细胞 (SMC) 主要表达 Akt1 亚型。一些研究小组已证明培养的 Akt1 缺失 EC 和 SMC 的迁移和增殖反应受损，从而支持 PI3K/Akt 信号传导在血管细胞类型中的促血管生成作用。 PI3K/Akt 信号传导在成人血管生成过程中也至关重要，因为 Akt1 的整体缺失会导致缺血和 VEGF 诱导的血管生成受损。 Akt 亚型的全局敲除模型有助于揭示 Akt 的生理作用。然而，非冗余表型的出现以及 Akt1 的普遍表达限制了我们以细胞特异性方式对 Akt1 功能的理解。因此，我们生成了条件 Akt1flox/flox 小鼠，以检查胚胎和出生后血管生成过程中 EC 和 SMC 特异性 Akt1 丢失的重要性。我们还在 Akt2 缺失的背景下培育了条件 Akt1 小鼠，有效地创建了诱导型双敲除小鼠，以解决可能的补偿效应。我们打算研究 EC 特异性 Akt1 缺失如何影响视网膜脉管系统和成人缺血诱导的血管生成。鉴于内皮通过 Akt 依赖性一氧化氮的产生来调节血管张力，我们将纳入血管反应性研究来测量对血管张力的后续影响。后肢缺血模型还将用于研究 SMC 特异性 Akt1 缺失如何影响由此产生的动脉生成/血管生成和血管重塑。这些研究将通过使用我们的诱导性双敲除小鼠来检查双 Akt1/2 缺失的后续影响得到进一步补充。因此，该提案的总体重点是使用几种已建立的分子、细胞和遗传方法来阐明 Akt1 和 Akt2 在两种主要血管细胞类型（EC 和 SMC）中的作用。