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

描述（由申请人提供）：血管化是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PI 3 K依赖性Akt活化促进血管生长。激活的Akt（Akt 1/Akt 2/Akt 3）靶向参与关键内皮功能的广泛底物，包括调节血管张力、血管生成和细胞向血管壁的募集。内皮细胞（EC）和血管平滑肌细胞（SMC）主要表达Akt 1亚型。几个研究小组已经证明了在培养的Akt 1-null EC和SMC中受损的迁移和增殖反应，从而支持PI 3 K/Akt信号在血管细胞类型中的促血管生成作用。PI 3 K/Akt信号传导在成人血管生成过程中也是至关重要的，因为Akt 1的整体丢失导致缺血和VEGF诱导的血管生成受损。Akt同种型的全局敲除模型有助于阐明Akt的生理作用。然而，非冗余表型的出现沿着Akt 1的普遍表达限制了我们对Akt 1细胞特异性功能的理解。因此，我们已经产生了条件Akt 1flox/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Akt 1丢失的重要性。我们还将条件性Akt 1小鼠与Akt 2-null背景进行了繁殖，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Akt 1缺失如何影响视网膜血管和成人缺血诱导的血管生成。鉴于内皮通过Akt依赖性一氧化氮产生调节血管张力，我们将纳入血管反应性研究，以测量对血管张力的后续影响。后肢缺血模型也将用于研究SMC特异性Akt 1丢失如何影响动脉生成/血管生成和血管重塑。这些研究将通过使用我们的诱导型双敲除小鼠来进一步补充，以检查双重Akt 1/2丢失的后续影响。因此，本提案的总体重点是阐明Akt 1和Akt 2在两种主要血管细胞类型（EC和SMC）中的作用，使用几种已建立的分子，细胞和遗传方法。

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.