Analysis of FAK and FIP200 in the cardiovascular system

描述(申请人提供)：拟议研究的长期目标是了解心血管系统发育和功能中的信号转导机制。上一期的研究主要集中在利用Cre-loxP介导的条件敲除(KO)技术对内皮细胞(ECs)和心肌细胞中的粘着斑激酶(FAK)进行条件性敲除，从而研究FAK在血管生成和血管生成中的作用以及在心脏发育和功能中的作用。对EC特异的FAK条件性KO小鼠的分析证实了FAK在体内胚胎发育过程中的血管生成和血管发育中的关键作用。此外，我们还发现，在分离的原代内皮细胞中，FAK的缺失导致了多种缺陷，包括细胞存活和迁移的减少，这可能是体内血管生成和血管发育缺陷的原因。对心肌细胞特异性FAK条件性KO小鼠的分析表明，FAK在心肌肥大的调节中发挥作用。此外，我们还为FIP200(200 kDa的FAK家族相互作用蛋白)构建了一个小鼠KO，以了解其在体内的功能。我们发现FIP200基因失活会导致因心力衰竭和肝脏损害而导致的妊娠中期/晚期胚胎死亡。对FIP200 KO小鼠的分析表明，TSC-mTOR和TNFa信号通路的改变导致心脏和肝脏细胞大小和细胞凋亡的变化可能是FIP200 KO小鼠发育异常和胚胎死亡的原因。尽管已有研究表明FAK和FIP200在心血管发育和功能中具有重要作用，但FAK和FIP200调控的特定信号通路在体内心血管发育中的作用尚未被研究。在我们先前和初步研究的基础上，我们建议1。利用分离的原代内皮细胞分析FAK信号通路在调控EC迁移、侵袭和血管生成中的作用。通过小鼠敲入法确定FAK信号通路在体内血管生成和血管发育中的作用，以及3.)采用细胞和分子生物学方法，结合各种相关KO小鼠的遗传交互作用研究，探讨FIP200对心脏发育过程中TSC-mTOR和TNFa信号通路的调控机制。相关性：这些研究将对FAK和FIP200在体内心血管系统中的信号转导机制产生重要的见解，并可能为心血管疾病新疗法的潜在开发提供关键信息。

DESCRIPTION (provided by applicant): The long term goal of the proposed studies is to understand the signal transduction mechanisms in the development and functions of the cardiovascular system. The previous funding period focused on the role of focal adhesion kinase (FAK) in endothelial cells (ECs) in vasculogenesis and angiogenesis as well as in cardiac myocytes in heart development and function by using Cre-loxP mediated conditional knockout (KO) of FAK in ECs and cardiomyocytes. Analysis of the EC-specific FAK conditional KO mice established a critical role for FAK in angiogenesis and vascular development during embryogenesis in vivo. Furthermore, we showed that deletion of FAK in the isolated primary ECs led to multiple defects including reduced cell survival and migration which may be responsible for the defective angiogenesis and vascular development in vivo. Analysis of the cardiomyocyte-specific FAK conditional KO mice suggested a role of FAK in the regulation of cardiac hypertrophy. In addition, we generated a mouse KO for FIP200 (FAK-family interacting protein of 200 kDa) to understand its functions in vivo. We found that inactivation of FIP200 gene resulted in mid/late-gestational embryonic lethality caused by heart failure and liver lesions. Analysis of the FIP200 KO mice suggested that alterations in TSC-mTOR and TNFa signaling pathways leading to changes in cell size and apoptosis in the heart and liver may be responsible for the developmental abnormality and embryonic lethality in FIP200 KO mice. Despite the progress suggesting important functions of FAK and FIP200 in the cardiovascular development and functions, the role of specific signaling pathways regulated by FAK and FIP200 has not been examined in the context of cardiovascular development in vivo. Based on our previous and preliminary studies, we propose to 1.) analyze the FAK signaling pathways in the regulation of EC migration, invasion and angiogenesis using isolated primary ECs, 2.) determine the role of FAK signaling pathways in angiogenesis and vascular development in vivo by using mouse knock-in approaches, and 3.) investigate the mechanisms of FIP200 regulation of TSC-mTOR and TNFa signaling pathways in cardiac development using a combination of cell and molecular biology approaches in isolated cardiomyocytes and genetic interaction studies of various relevant KO mice. Relevance: These studies will generate significant insights into the mechanisms of signal transduction by FAK and FIP200 in the cardiovascular system in vivo and may also provide critical information for potential development of novel therapies for cardiovascular diseases.