Mechanisms Regulating Vascular Homeostasis

项目摘要 正常内皮细胞（EC）功能的调节在血管稳态和血管内皮细胞的生长中起着关键作用。 预防心血管疾病（CVD）。心血管疾病中的内皮功能障碍的特征是血管内皮细胞的丧失。 内皮型一氧化氮合酶（eNOS）衍生的一氧化氮（NO）。Tie受体及其配体， 血管生成素（angiopoietins，Ang）是血管稳态和EC从静止过渡的重要调节因子 到“激活”和血管重塑。而Ang-1/Tie 2信号转导促进EC存活和静止， 部分通过激活Akt和eNOS，同源Tie 1受体已显示促进EC 活化、炎症和动脉粥样硬化。然而，Tie 2介导的血管生成也需要Tie 1， 和Tie 1的整体功能效应似乎是依赖于上下文的，并在一定程度上与 炎症与Tie 2不同，Tie 1没有已知的激活配体，因此它的信号传导途径和 作用机制一直难以研究，而且仍然知之甚少。我们找到了一本小说， 以前未表征的分子支架蛋白，Caskin 2，作为Tie 1相互作用蛋白。尽管很少 研究已经专门针对Caskin 2的功能，它已被确定为EC的强标记物， 许多分子研究已经将Caskin 2与血管缺陷相关的表型联系起来， 体内平衡重要的是，我们的研究表明，Caskin 2是Tie受体之间的关键分子联系， 信号传导、eNOS生物学和血管稳态。我们对这一建议的初步研究表明， Caskin 2在体外和体内的EC中高度表达，并且Caskin 2促进EC静止和存活 体外Caskin 2与Tie 1的结合被Tie 1上T794的磷酸化破坏，这是一个由Tie 1介导的事件。 PAK以Rac依赖的方式存在，并且是血管生成所需的。此外，Caskin 2是血管生成所必需的。 1/Tie 2通过Akt介导的信号传导，表明Caskin 2整合了来自Tie 1和Tie 2的信号， 调节血管内环境稳定。此外，Caskin 2结合eNOS及其激活剂钙调蛋白（CaM）， 增加eNOS表达和活性，并通过流体剪切应力和KLF 2上调。最后我们 显示Caskin 2-/-小鼠具有增加血管渗透性、动脉粥样硬化、病理性血管生成 并且是高血压的，所有的血管内稳态紊乱都以异常eNOS信号传导为特征。基于 基于这些发现，我们假设Caskin 2是Tie受体信号传导的关键整合子， 部分通过eNOS激活调节血管稳态。为了验证这一假设， 本提案的目的是：1）确定Caskin 2调节Tie的分子机制 受体介导的内皮功能和eNOS活性; 2）确定Caskin 2 响应并感测流体剪切应力的变化，以在体外调节EC表型;以及3）确定 Caskin 2缺乏对体内血管稳态的影响。

PROJECT SUMMARY Regulation of normal endothelial cell (EC) function plays a critical role in vascular homeostasis and the prevention of cardiovascular disease (CVD). Endothelial dysfunction in CVD is characterized by loss of endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO). The Tie receptors and their ligands, the angiopoietins (Ang), are important regulators of vascular homeostasis and the transition from EC quiescence to "activation" and vascular remodeling. Whereas Ang-1/Tie2 signaling promotes EC survival and quiescence, in part through activation of Akt and eNOS, the homologous Tie1 receptor has been shown to promote EC activation, inflammation, and atherosclerosis. However, Tie1 is also required for Tie2-mediated angiogenesis, and Tie1's overall functional effects appear to be context-dependent and linked, in part, to the presence of inflammation. Unlike Tie2, Tie1 has no known activating ligands, therefore its signaling pathways and mechanisms of action have been difficult to study and remain poorly understood. We identified a novel and previously uncharacterized molecular scaffolding protein, Caskin2, as a Tie1-interacting protein. Although few studies have specifically addressed Caskin2's function, it has been identified as a strong marker of ECs, and numerous molecular studies have linked Caskin2 to phenotypes associated with defects in vascular homeostasis. Importantly, our studies indicate that Caskin2 is a critical molecular link between Tie receptor signaling, eNOS biology, and vascular homeostasis. Our preliminary studies in this proposal demonstrate that Caskin2 is highly expressed in ECs in vitro and in vivo and that Caskin2 promotes EC quiescence and survival in vitro. Caskin2 binding to Tie1 is disrupted by phosphorylation of T794 on Tie1, an event that is mediated by PAK in a Rac-dependent manner and that is required for angiogenesis. Moreover, Caskin2 is required for Ang- 1/Tie2-mediated signaling through Akt, suggesting that Caskin2 integrates signals from both Tie1 and Tie2 to regulate vascular homeostasis. Furthermore, Caskin2 binds eNOS and its activator calmodulin (CaM) and increases eNOS expression and activity and is upregulated by fluid shear stress and by KLF2. Finally, we show that Caskin2–/– mice have increased vascular permeability, atherosclerosis, pathological angiogenesis, and are hypertensive, all disorders of vascular homeostasis characterized by abnormal eNOS signaling. Based on these findings, we hypothesize that Caskin2 is a critical integrator of Tie receptor signaling that regulates vascular homeostasis in part through eNOS activation. To test this hypothesis, the Specific Aims of this proposal are to: 1) Determine the molecular mechanisms by which Caskin2 regulates Tie receptor-mediated endothelial function and eNOS activity; 2) Determine the mechanisms by which Caskin2 responds to and senses changes in fluid shear stress to regulate EC phenotypes in vitro; and 3) Determine the effects of Caskin2 deficiency on vascular homeostasis in vivo.