Regulation of Akt-integrin pathway in endothelial function and neovasculature

描述（由申请人提供）：尽管在血管生物学和发育方面取得了相当大的进展，但仍缺乏保护内皮功能的技术，并且在了解如何形成稳定和成熟的新血管方面存在缺陷。这些研究的主要目标是剖析详细的分子机制，并确定新的靶点调节内皮功能和新血管生成。这是我们项目的重新提交，该项目集中于整合素-Akt信号轴在血管生物学中的作用。我们已经证明，内皮细胞上的整合素活化是血管生成和血管稳态所必需的，并确定了调控这一过程的关键分子。从机制上讲，我们已经确定整合素/Akt轴作为血管生长和成熟的重要驱动因素，这表明该途径可以作为组织特异性血管生成的开/关开关。我们的初步数据表明，Akt在内皮细胞控制血管功能和血管生成。我们将重点关注Akt在内皮细胞迁移和整合素功能中的作用，并确定控制这种相互作用的分子相互作用。主要假设是：内皮Akt 1通过PINCH、ILK、Parvin的异源三聚体复合物被募集到整合素，并作为整合素由内而外信号传导的重要调节剂，其反过来控制体内内皮迁移和血管生成。虽然其他平行途径可能在Akt不存在的情况下介导细胞存活和增殖，但它对于内皮附着和迁移是不可或缺的。提出了以下具体目标：目标1。为了建立血管生成中内皮Akt在整合素依赖性功能中的作用：我们将确定Akt活性水平，其足以在血管生成反应期间支持体内整合素功能。我们将评估内皮细胞Akt缺失如何以及为什么会影响新生血管的关键特征。目标2.确定EC中整合素由内向外与由外向内信号传导所需的Akt活性和Akt亚型水平。我们将比较单个Akt同种型在由VEGFR或由TLR 2活化触发的整联蛋白由内而外信号传导的调节中的直接参与（作为内皮中新的VEGF非依赖性途径的实例）。将使用组成型活性整合素或强制整合素活化进行补救实验。目标3.目的探讨Akt 1募集整合素复合物的分子机制及其在内皮细胞整合素功能中的作用。我们将重点关注Akt 1的直接招募整合素相关复合物（ILK/ PINCH/Parvin和Kindlin），并确定该复合物如何协调整合素在内皮上的功能。为此，我们将确定该复合物的结构决定因素（Akt 1内与Parvin相互作用所需的残基，这是整个复合物形成的先决条件），并显示个体相互作用的破坏如何影响双向整合素信号传导。总之，我们的研究将显示Akt-整合素轴如何在复杂的体内环境中调节血管稳态和血管生成，并揭示连接Akt信号传导与整合素功能的分子决定因素。这些研究将确定健康和疾病中内皮功能的新细胞和分子机制。

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.