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

描述(申请人提供)：血管形成是胚胎发育和血管重塑的重要过程。多种刺激(如切应力、细胞因子、生长因子)通过依赖PI3K激活Akt促进血管生长。激活的Akt(Akt1/Akt2/AKT3)靶向广泛的底物，参与关键的内皮功能，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Akt1亚型。一些研究小组已经证明Akt1缺失的EC和SMC的迁移和增殖反应受损，从而支持PI3K/Akt信号在血管细胞类型中的促血管生成作用。PI3K/Akt信号在成人血管生成过程中也是至关重要的，因为Akt1的整体缺失会导致缺血和血管内皮生长因子诱导的血管生成受损。Akt亚型的全球敲除模型帮助解开了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的方式理解Akt1的功能。因此，我们建立了条件性Akt1Flox/FLOX小鼠，以检测EC和SMC特异性Akt1丢失在胚胎和出生后血管生成过程中的重要性。我们还将有条件的Akt1小鼠培育成Akt2缺失的背景，有效地创造了一个可诱导的双基因敲除小鼠，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血管系统和成人缺血诱导的血管生成。鉴于内皮通过Akt依赖的一氧化氮的产生来调节血管张力，我们将包括血管反应性研究，以衡量由此对血管张力的影响。后肢缺血模型也将被用于研究SMC特异性Akt1的缺失如何影响由此产生的动脉生成/血管生成和血管重塑。这些研究将与我们的可诱导双基因敲除小鼠进一步补充，以检验双重Akt1/2缺失的后续影响。因此，这项建议的总体重点是阐明Akt1和Akt2在两种主要的血管细胞类型(EC和SMC)中的作用，使用几种已建立的分子、细胞和遗传学方法。

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.