Regulation of Akt-integrin pathway in endothelial function and neovasculature

描述(申请人提供)：尽管在血管生物学和发育方面取得了长足的进步，但缺乏保护内皮功能的技术，以及对稳定和成熟的新血管是如何形成的了解不足。这些研究的主要目的是剖析详细的分子机制，并确定调节内皮功能和新血管生成的新靶点。这是我们专注于整合素-Akt信号轴在血管生物学中的作用的项目的重新提交。我们已经证明了内皮上整合素的激活对于血管生成和血管内稳态是必不可少的，并且已经确定了调节这一过程的关键分子。从机制上讲，我们已经确定整合素/Akt轴是血管生长和成熟的重要驱动因素，这表明这一途径可以作为组织特异性血管生成的开关。我们的初步数据表明，内皮细胞中的Akt调控血管功能和血管生成。我们将重点研究Akt在内皮迁移和整合素功能中的作用，并将确定控制这一相互作用的分子相互作用。主要的假说是：内皮细胞Akt1通过Pinch、ilk、Parvin的异源三聚体复合体被招募到整合素，并作为整合素内向外信号的重要调节因子，进而控制体内的内皮细胞迁移和血管生成。虽然在没有Akt的情况下，其他平行通路可能介导细胞的存活和增殖，但它对内皮细胞的附着和迁移是不可或缺的。目的：1.确定内皮细胞Akt在血管生成中整合素依赖功能中的作用：我们将确定在血管生成反应过程中足以支持体内整合素功能的Akt活性水平。我们将评估内皮细胞Akt缺失如何以及为什么影响新生血管的关键特征。目的2.确定EC中整合素由内向外与由外向内信号转导所需的Akt活性和Akt亚型水平。我们将比较单个Akt亚型在VEGFR或TLR2激活(作为内皮中一种新的血管内皮生长因子非依赖性通路的一个例子)触发的整合素内向外信号调节中的直接参与。将进行具有结构性活性的整合素或强制激活整合素的救援实验。目的3.建立Akt1募集到整合素复合体的详细分子机制以及该复合体在内皮细胞整合素功能中的作用。我们将集中于Akt1与整合素相关复合体(ILK/Pinch/Parvin和Kindlin)的直接募集，并确定该复合体如何协调内皮上的整合素功能。为此，我们将确定该复合体的结构决定因素(与Parvin相互作用所需的Akt1内残基，这是形成整个复合体的先决条件)，并展示单个相互作用的中断如何影响双向整合素信号。总之，我们的研究将揭示Akt-整合素轴如何在复杂的体内环境中调节血管内稳态和血管生成，并揭示将Akt信号与整合素功能联系起来的分子决定因素。这些研究将确定健康和疾病中潜在的内皮功能的新的细胞和分子机制。

DESCRIPTION (provided by applicant): Despite the considerable progress in vascular biology and development, there is a lack of technologies preserving endothelial function, as well as deficits in understanding how stable and mature neovasculature is formed. The main goal of these studies is to dissect the detailed molecular mechanisms and identify novel targets regulating endothelial function and neoangiogenesis. This is a resubmission of our project focused on the role of integrin-Akt signaling axis in vascular biology. We have demonstrated that integrin activation on endothelium is essential for angiogenesis and vascular homeostasis and have identified key molecules regulating this process. Mechanistically, we have identified integrin/Akt axis as an important driver of vascular growth and maturation, suggesting that this pathway can act as an on/off switch for tissue specific angiogenesis. Our preliminary data demonstrate that Akt in endothelium controls vascular functions and angiogenesis. We will focus on the role of Akt in endothelial migration and integrin function and will determine molecular interactions controlling this interplay. The main hypothesis is: endothelial Akt1 is recruited to integrins via heterotrimeric complex of PINCH, ILK, Parvin, and serves as an essential regulator of integrin inside-out signaling, which, in turn, controls endothelial migratio and angiogenesis in vivo. While other parallel pathways might mediate cell survival and proliferation in the absence of Akt, it is indispensable for the endothelial attachment and migration. The following specific Aims are proposed: Aim 1. To establish the role for endothelial Akt in integrin-dependent functions in angiogenesis: We will determine the levels of Akt activity which are sufficient to support integrin functions in vivo during angiogenic responses. We will assess how and why Akt deletion in endothelium affects the key characteristics of neovasculature. Aim 2. To determine the levels of Akt activity and Akt isoforms required for integrin inside-out vs. outside-in signaling in EC. We will compare the direct involvement of individual Akt isoforms in the regulation of integrin inside-out signaling triggered either by VEGFR or by TLR2 activation (as an example of a novel VEGF- independent pathway in endothelium). Rescue experiments with constitutively active integrins or forced integrin activation will be performed. Aim 3. To establish a detailed molecular mechanism of Akt1 recruitment to integrin complex and the role for this complex in integrin function in endothelium. We will focus on the direct recruitment of Akt1 to integrin-associated complex (ILK/ PINCH/Parvin and Kindlin) and determine how this complex coordinates integrin functions on endothelium. To this end, we will determine the structural determinants of this complex (residues within Akt1 required to interact with Parvin, which is a prerequisite for the formation o the entire complex) and show how disruption of the individual interactions affects bidirectional integrin signaling. Together, our studies will show how the Akt-integrin axis regulates vascular homeostasis and angiogenesis in a complexity of in vivo settings, and reveal the molecular determinants connecting Akt signaling to integrin function. These studies will identify novel cellular and molecular mechanisms underlying endothelial functions in health and disease.