Mechanisms Driving Apoptosis Resistance in Pulmonary Hypertension

项目摘要 尽管有可用的治疗方法，肺动脉高压（PAH）仍然是一种致死性诊断。PAH是 其特征在于肺血管系统的广泛重塑，涉及血管闭塞性血管炎的形成。 病变和增厚的血管壁中层，两者都含有肺动脉平滑肌 细胞（PASMCs）。已经证明，从建立良好的PAH大鼠模型中分离的PASMCs， 在基础条件和刺激条件下均抗凋亡。水通道蛋白1 （AQP 1）最初被描述为水运输通道，但最近已被牵连在其他 细胞功能，包括迁移和增殖，在几种不同的癌症类型中， 具有抗凋亡性。AQP 1在从PAH大鼠模型分离的PASMCs中上调，表明“准 尽管AQP 1在细胞凋亡抵抗中的确切作用尚不清楚，令人兴奋的新数据来自 一项未发表的使用肺裂解物的蛋白质组学研究表明， caspase-3是一种在细胞凋亡中被激活的酶蛋白，它被转运到细胞核，在那里它启动细胞凋亡。 死亡在利用生物素连接酶技术的邻近研究中，我已经证明了AQP 1和总半胱天冬酶- 3在活细胞中非常接近。此外，AQP 1蛋白的计算机分析揭示了3种潜在的 半胱天冬酶-3切割位点，其提供了这种蛋白质-蛋白质相互作用的机制。这些数据一起 表明AQP 1与caspase-3相互作用，提供了AQP 1、caspase-3 级联和抗凋亡。这个应用程序提供了一个培训工具，因为我探索一个潜在的 AQP 1在PAH期间调节细胞凋亡的机制。目标1旨在确定 AQP 1上的胞浆caspase-3切割位点是AQP 1/caspase-3相互作用所必需的，Aim 2用于 评估AQP 1对capsase-3核定位的影响，如果增加，最终将建立Aim 3 AQP 1是必需的和/或足以赋予细胞凋亡抗性。用于实现这些目标的技术 包括但不限于蛋白质表达和定点诱变，生物素连接酶邻近测定， 免疫共沉淀、PAH动物模型和原代细胞分离、免疫荧光和共聚焦显微镜 显微镜、核/胞质分级分离、发光半胱天冬酶-3/7活性测定、Hoechst染色和 TUNEL染色。该项目的完成将为AQP 1和AQP 2之间的相互作用提供新的见解。 caspase-3和AQP 1在细胞凋亡抵抗中的作用，并为新的治疗方法提供新的途径。 目标的在设计和执行本研究中获得的技能和实验结果将提供 K奖的必要基础和一个很好的平台，在这个平台上开始独立的职业生涯 资助的临床科学家专注于PAH。从这项工作中获得的见解也可能产生超越 PAH与细胞对凋亡的抵抗对疾病发展至关重要的任何诊断有关。

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) remains a fatal diagnosis despite available therapies. PAH is characterized by extensive remodeling of the pulmonary vasculature involving the formation of vaso-occlusive lesions and a thickened medial layer of the vascular wall, both of which contain pulmonary arterial smooth muscle cells (PASMCs). It has been demonstrated that PASMCs isolated from a well-established rat model of PAH are resistant to apoptosis under both basal and stimulated conditions. The cell membrane protein aquaporin 1 (AQP1) was initially described as a water transport channel, but more recently has been implicated in other cellular functions including migration and proliferation, and in several distinct cancer types, has been associated with apoptosis resistance. AQP1 is upregulated in PASMCs isolated from rat models of PAH suggesting a ‘quasi- malignant’ disease model, although AQP1’s exact role in apoptosis resistance is unclear. Exciting new data from an unpublished proteomics study using lung lysates demonstrates that AQP1 immunoprecipitates with total caspase-3, a enzymatic protein activated in apoptosis which is translocated to the nucleus where it initiates cell death. In proximity studies utilizing biotin ligase techniques, I have demonstrated that AQP1 and total caspase- 3 come within close proximity in live cells. Furthermore, in silico analysis of the AQP1 protein reveals 3 potential caspase-3 cleavage sites, which provide a mechanism for this protein-protein interaction. Together, these data suggest that AQP1 interacts with caspase-3, providing a novel relationship between AQP1, the caspase cascade, and resistance to apoptosis. This application serves to provide a training vehicle as I explore a potential mechanism by which AQP1 regulates apoptosis during PAH. Aim 1 is designed to determine whether the cytosolic caspase-3 cleavage site(s) on AQP1 are necessary for AQP1/caspase-3 interaction, Aim 2 serves to evaluate the impact of AQP1 on nuclear localization of capsase-3, and finally Aim 3 will establish if increased AQP1 is necessary and/or sufficient to confer apoptosis resistance. Techniques utilized to address these aims include but are not limited to protein expression and site directed mutagenesis, biotin ligase proximity assays, co-immunoprecipitation, animal models of PAH and primary cell isolation, immunofluorescence and confocal microscopy, nuclear/cytosolic fractionation, luminescent caspase-3/7 activity assay, Hoechst staining and TUNEL staining. Completion of this project will provide novel insight into the interaction between AQP1 and caspase-3 and the role for AQP1 in apoptosis resistance as well as provide a novel pathway for new therapeutic targets. The skills obtained in the design and execution of this study and the experimental results will provide the necessary foundation for a K award and an excellent platform on which to start a career as an independently funded clinician scientist focused on PAH. Insights gained from this work also could have implications beyond PAH to any diagnosis in which cellular resistance to apoptosis is essential to the development of disease.