Mechanisms Regulating Vascular Homeostasis

项目总结 正常内皮细胞(EC)功能的调节在血管内稳态中起着至关重要的作用 预防心血管疾病(CVD)。血管内皮细胞功能障碍的特征是血管内皮功能丧失。 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，即 血管生成素(Angiopoietins，Ang)是血管内皮细胞内稳态和从静息状态过渡的重要调节因子 到“激活”和血管重塑。而Ang-1/Tie2信号促进EC存活和静止， 部分通过激活Akt和eNOS，同源的Tie1受体已被证明促进EC 活化、炎症和动脉粥样硬化。然而，Tie1也是Tie2介导的血管生成所必需的， 和S的总体功能效应似乎是上下文相关的，并部分地与存在 发炎。与Tie2不同，Tie1没有已知的激活配体，因此它的信号通路和 行动机制一直很难研究，人们对此也知之甚少。我们确定了一部小说， 以前未知的分子支架蛋白，Caskin2，作为一种Tie1相互作用蛋白。虽然为数不多 已有研究明确了Caskin2的S功能，它已被确定为内皮细胞的一个强有力的标志，并且 许多分子研究已将Caskin2与与血管缺陷相关的表型联系起来 动态平衡。重要的是，我们的研究表明，Caskin2是Tie受体之间的关键分子联系 信号、eNOS生物学和血管动态平衡。我们在这项建议中的初步研究表明 Caskin2在体外和体内高表达于内皮细胞，并促进内皮细胞的静止和存活 在试管中。Caskin2与Tie1的结合被Tie1上T794的磷酸化破坏，这一事件是由 PAK是一种RAC依赖的方式，是血管生成所必需的。此外，Ang-2还需要Caskin2。 1/Tie2通过Akt介导的信号传递，表明Caskin2整合了来自Tie1和Tie2的信号 调节血管动态平衡。此外，Caskin2还与eNOS及其激活剂钙调蛋白(CaM)结合，并 增加eNOS的表达和活性，并被流体切应力和KLF2上调。最后，我们 显示Caskin2-/-小鼠血管通透性增加，动脉粥样硬化，病理性血管生成， 且均为高血压，均为以eNOS信号异常为特征的血管内稳态紊乱。基座 根据这些发现，我们假设Caskin2是Tie受体信号的关键整合者 部分通过eNOS的激活来调节血管的动态平衡。为了检验这一假设，具体的 这项提议的目的是：1)确定Caskin2调节Tie的分子机制 受体介导的内皮功能和eNOS活性；2)决定Caskin2的机制 在体外对流体剪切力的变化作出反应并感知，以调节EC的表型；以及3)确定 Caskin2缺乏对体内血管动态平衡的影响。

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.