Regulation of endothelial gene expression by cerebral cavernous malformation complex

项目摘要/摘要 导师职业发展奖将给我机会接受额外的培训 和研究经验，以实现我获得独立教员的长期目标 在研究型大学任职，并在心血管生物学领域做出贡献。这个 提议的项目是一个宝贵的工具，可以扩展我在复杂研究中的知识和专长 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 在多学科和高度协作的环境中进行研究，方法是与 从临床医学到结构和分子生物学。此外，金斯伯格的实验室也很好 配备细胞、蛋白质组和分子生物学工具，以了解细胞和分子 脑海绵状血管畸形(CCM)相关基因蛋白产物的生物学(KRIT1， Krev-相互作用陷阱-1、CCM2和PDCD10)。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丢失，细胞培养模型 建立了以时间控制的方式删除小鼠内皮细胞中Krit1的方法。为此，转基因 携带Krit1(Krit1fl/fl)和内皮特异性三苯氧胺调节的Cre的小鼠 重组酶(pdgfb-iCreERT2)。在初步研究中，表达增加的 转录因子KLF2参与Krit1对斑马鱼心脏的影响 血栓反应素1(TSP1)是一种抗血管生成蛋白，发生、发展和表达减少 观察到其拮抗血管内皮生长因子信号转导通路。将进行功能增益实验，以 评估TSP1的重新表达是否可以逆转KRIT1的丢失影响内皮表型和 功能。此外，这将通过调查TSP1基因转录是否被 在培养细胞中由于Krit1基因的缺失而被抑制。由于SP1/KLF结合位点在 人和小鼠TSP1启动子区域的确定，KLF2在TSP1下调中的作用 作为对Krit1基因失活的反应，将对其进行调查。此外，斑马鱼CCM模型 将被用来评估TSP1在体内心血管畸形过程中丢失的影响。这个 这份建议书中描述的研究和加州大学圣迭戈分校的环境将完成我在综合体的培训 研究方法和对KRIT1基因功能的基本问题的洞察 在内皮细胞和生物体水平上。

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.