Analysis of intracellular signaling in angiogenesis

描述（由申请人提供）：拟议研究的长期目标是了解控制心血管系统发育和功能的信号转导机制。在之前的资助期内，我们重点研究了胚胎血管生成过程中ECs中focal adhesion kinase （FAK）信号通路的作用和机制，以及FAK- 200 kDa家族相互作用蛋白（FIP200）在心脏发育中的作用。利用基于分离的原代无FAK的EC的功能重构方法，我们发现FAK的S732磷酸化在有丝分裂过程中通过调节中心体功能促进EC增殖和血管生成中的新作用。我们还创建了两种不同的FAK突变敲入小鼠模型。使用EC特异性FAK激酶缺陷（KD）突变敲入小鼠模型，我们证明了FAK在胚胎发育期间分别在EC存活和屏障功能中激酶独立和依赖的作用。除了这些和其他几项已发表的研究表明FAK和FIP200在心脏发育中的作用外，该资助还支持我们关于FIP200在胎儿造血干细胞中的作用和机制的研究。在初步研究中，我们建立了新的小鼠模型，诱导ec特异性FAK敲除（KO）和KD突变敲除小鼠，并显示FAK及其激酶活性在成人血管生成中的作用。此外，我们观察到在FAK缺失或其激酶活性丧失时Notch信号增加，这表明FAK通过关键的Notch通路调节血管生成的潜在新机制。我们还生成并分析了胚胎和诱导的ec特异性TSC1（结节硬化复合体1）的KO，这为TSC/mTOR信号在小鼠体内模型中成年生物胚胎血管发育和血管生成中的作用提供了第一个直接证据。尽管取得了这些进展，但对于FAK信号在成人体内血管生成调控中的作用和机制，我们仍然知之甚少。同样，胚胎和成人血管生成过程中内皮细胞中TSC/mTOR信号传导的机制尚未在体内直接评估。基于我们之前和初步的研究，我们建议：1)。通过诱导ec特异性FAK KO和KD突变敲入小鼠模型，分析FAK及其激酶活性在出生后血管生成中调控Notch信号通路中的作用和机制，2)。通过ec特异性TSC1 KO小鼠模型研究TSC/mTOR信号在血管发育和血管生成中的作用和机制；研究TSC/mTOR信号通路在出生后血管生成中的作用、机制及其对VEGF表达的调控。这些研究将对体内血管生成调控中的细胞内信号传导机制产生重要的见解，也可能为血管生成相关疾病的新疗法的潜在开发提供重要信息。

DESCRIPTION (provided by applicant): The long-term goal of the proposed studies is to understand the signal transduction mechanisms governing the development and function of the cardiovascular system. In the previous funding period, we focused on the role and mechanisms of focal adhesion kinase (FAK) signaling in ECs during embryonic angiogenesis and FAK- family interacting protein of 200 kDa (FIP200) in cardiac development. Using a functional reconstitution approach based on isolated primary FAK-null ECs, we identified a novel role for S732 phosphorylation of FAK in promoting EC proliferation and angiogenesis by the regulation of centrosome functions during mitosis. We have also created two different FAK mutant knockin mouse models. Using the EC-specific FAK kinase- defective (KD) mutant knockin mouse model, we demonstrated the role of kinase-independent and -dependent functions of FAK in EC survival and barrier function, respectively, during embryonic development. In addition to these and several others published studies that show the role of FAK and FIP200 in cardiac development, this grant also supported our studies on the role and mechanisms of FIP200 in fetal hematopoietic stem cells. In preliminary studies, we established new mouse models with the inducible EC-specific FAK knockout (KO) and KD mutant knockin mice and showed a role of FAK and its kinase activity in adult angiogenesis. Moreover, we observed increased Notch signaling upon FAK deletion or loss of its kinase activity, suggesting a potentially novel mechanism of FAK in the regulation of angiogenesis through the critical Notch pathway. We also generated and analyzed both embryonic and inducible EC-specific KO of TSC1 (tuberous sclerosis complex 1), which provided the first direct evidence for a role of TSC/mTOR signaling in the embryonic vascular development and angiogenesis of adult organisms in mouse models in vivo. Despite these progresses, still relatively little is known about the role and mechanisms o FAK signaling in the regulation of angiogenesis in adult organism in vivo. Likewise, the mechanisms of TSC/mTOR signaling in ECs during embryonic and adult angiogenesis have not been assessed directly in vivo. Based on our previous and preliminary studies, we propose to 1). Analyze the role and mechanism of FAK and its kinase activity in the regulation of Notch signaling in postnatal angiogenesis by using inducible EC-specific FAK KO and KD mutant knockin mouse models, 2). Investigate the role and mechanisms of TSC/mTOR signaling in vascular development and angiogenesis by using an EC-specific TSC1 KO mouse model, and 3). Study the role and mechanisms of TSC/mTOR signaling and its regulation of VEGF expression in postnatal angiogenesis. These studies will generate significant insights into the mechanisms of intracellular signaling in the regulation of angiogenesis in vivo and may also provide critical information for potential development of novel therapies for angiogenesis related diseases.