Regulation of endothelial gene expression by cerebral cavernous malformation complex

项目概要/摘要 导师职业发展奖将使我有机会接受额外的培训 和必要的研究经验，以实现我的长期目标，获得一个独立的教师 在一所研究型大学的职位，并有助于心血管生物学领域。的 建议的项目是一个有价值的工具，以扩大我的知识和专业知识，在复杂的研究 方法，包括小鼠和斑马鱼遗传学/分子生物学，应用于 心血管领域加州大学圣地亚哥分校（UCSD）医学院 通过与同事互动，在多学科和高度协作的环境中进行研究， 从临床医学到结构和分子生物学。此外，金斯伯格实验室 配备了细胞，蛋白质组学和分子生物学工具，以了解细胞和分子 涉及脑海绵状畸形（CCM）的基因的蛋白产物的生物学（KRIT 1， Krev-Interaction Trapped-1、CCM 2和PDCD 10）。为了确定主要的分子过程 参与Krit1诱导的内皮细胞表型和功能改变的损失， 以时间控制的方式在小鼠内皮中缺失Krit1。为此，转基因 携带Krit1 floxed等位基因（Krit1fl/fl）和内皮特异性他莫昔芬调节Cre的小鼠 重组酶（Pdgfb-iCreERT2）。在初步研究中， Kruppel样因子2（KLF2），一种与Krit1对斑马鱼心脏作用有关的转录因子 血栓反应蛋白1（TSP1），一种抗血管生成蛋白， 拮抗VEGF信号传导。将进行功能增益实验， 评估TSP1的再表达是否可以逆转KRIT 1的损失影响内皮表型， 功能此外，这将通过调查TSP1基因转录是否是 在培养的细胞中由于Krit1的缺失而被抑制。由于SP1/KLF结合位点在 人和小鼠TSP1启动子区的鉴定，KLF2在TSP1下调中的作用， 对Krit1基因失活的反应。此外，斑马鱼CCM模型 将用于评估体内心血管畸形期间Tsp1丢失的影响。的 本建议中描述的研究和UCSD的环境将完成我在复杂的 研究方法，并提供有关KRIT 1基因功能的基本问题的见解 在内皮细胞和生物体水平。

Project Summary/Abstract The Mentored Career Development Award will give me the opportunity to receive the additional training and research experience necessary to achieve my long-term goal of obtaining an independent faculty position at a research-oriented university, and contribute to the field of Cardiovascular Biology. The proposed project is a valuable tool to extent my knowledge and expertise in complex research methods, including mouse and zebrafish genetics/molecular biology, that are applied in the cardiovascular field. The University of California, San Diego (UCSD) School of Medicine conducts research in a multidisciplinary and highly collaborative environment by interacting with colleagues that span clinical medicine to structural and molecular biology. Moreover, the Ginsberg laboratory is well equipped with cellular, proteomic, and molecular biology tools to understand cellular and molecular biology of the protein products of genes implicated in cerebral cavernous malformations (CCM) (KRIT1, Krev- Interaction Trapped-1, CCM2, and PDCD10). In order to identify the major molecular processes involved in loss of Krit1-induced altered endothelial phenotype and function, a cell culture model to delete Krit1 in mouse endothelium in a time-controlled manner was established. To this end, transgenic mice bearing floxed alleles of Krit1 (Krit1fl/fl) and an endothelial-specific tamoxifen-regulated Cre recombinase (Pdgfb-iCreERT2) were used. In preliminary studies, an increase in expression of Kruppel-like factor 2 (KLF2), a transcription factor implicated in the effects of Krit1 on zebrafish heart development, and a decrease in expression of Thrombospondin 1(TSP1), an anti-angiogenic protein that antagonizes VEGF signaling, was observed. Gain-of-function experiments will be performed to assess whether re-expression of TSP1 can reverse loss of KRIT1 affects endothelial phenotype and function. Moreover, this will be extended by investigating whether TSP1 gene transcription is suppressed as a consequence of deletion of Krit1 in cultured cells. Since SP1/KLF binding sites in human and mouse TSP1 promoter region were identified, the role of KLF2 in downregulation of TSP1 in response to genetic inactivation of Krit1 will be investigated. In addition, the zebrafish CCM model will be used to assess the impact of loss of Tsp1 during cardiovascular malformations in vivo. The studies described in this proposal and the environment at UCSD will complete my training in complex research methods and provide insight into fundamental questions about the function of the KRIT1 gene at the endothelial cell and organism level.