Mechanisms of Thrombospondin-1 as a pulmonary vascular mediator

Thrombospondin-1 作为肺血管介质的机制

• 批准号: 9537762
• 负责人: Carla F. Kim
• 金额: $ 12.1万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-15 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9537762
• 关键词: Acute; Adult; Alveolar; Alveolar Cell; Angiogenic Factor; BMP4; Biological Assay; Blood Vessels; Bronchopulmonary Dysplasia; CD47 gene; Calcineurin; Cell Proliferation; Cell model; Cells; Chronic lung disease; Coculture Techniques; Coupled; Data; Development; Dimensions; Disease; Endothelial Cells; Ensure; Epithelial Cells; Exposure to; Growth; Homeostasis; Hyperoxia; Impairment; In Vitro; Injury; Interruption; Knockout Mice; Life; Ligands; Lung; Lung diseases; Mediating; Mediator of activation protein; Modeling; Mus; Neonatal; Neonatal Hyperoxic Injury; Organoids; Outcome; Pathway interactions; Play; Premature Infant; Premature Infant Diseases; Prevention; Process; Proteins; Recombinants; Regulation; Role; Signal Transduction; Stem cells; System; Testing; Therapeutic Intervention; Thrombospondin 1; Thrombospondins; Tube; WNT Signaling Pathway; Work; cell type; in vivo; innovation; insight; lung development; lung injury; lung vascular injury; migration; novel; novel therapeutic intervention; prevent; receptor; repaired; response; therapeutic development; transcription factor; vasculogenesis

Summary Endothelial cells play a critical role in multiple aspects of lung development and homeostasis, yet the precise mechanisms that regulate vasculogenesis and how endothelial cells signal to other cell types to orchestrate lung development have not been well defined. Accurate regulation of these processes is critical to ensure proper lung development and prevent bronchopulmonary dysplasia (BPD), a lung disease of premature infants that often results in life-long complications. Hallmarks of BPD are impaired vasculature and arrested lung development. We created three-dimensional (3D) co-culture systems in which lung endothelial cells (LuMECs) drive the putative lung stem cells, BASCs, to produce bronchiolar or alveolar organoids. These organoid cultures provide an opportunity to model interactions that occur between endothelial cells and alveolar cells during lung development. Using this system, we discovered a novel BMP4-NFATc1- Thrombospondin (TSP1) pathway in endothelial and epithelial cells in the adult lung. We showed that TSP1, known as an anti-angiogenic factor, is required for the development of alveolar organoids. We also identified R-spondin2 (Rspo2), which regulates Wnt signaling in other systems, as a BMP4-regulated protein that may be downstream of TSP1 in endothelial cells. The mechanisms underlying TSP1 regulation of lung vascular development and alveolarization are unknown. We hypothesize that endothelial-derived TSP1 induced by BMP4-NFATc1 signaling inhibits pulmonary vascular development and is required for proper lung development. Further, we posit that vascular lung injury, such as that seen with hyperoxia exposure, impairs TSP1 signaling, thereby altering vasculogensis and blocking alveolar development. In Aim 1, we will determine how TSP1- CD47 interactions regulate vasculogenesis and lung development. Defective lung endothelial cells from mice exposed to hyperoxia will be used in lung organoid co-cultures to test the impact of impaired endothelial cell signals on alveolarization. Similarly, cells from knockout mice will be used in neonatal lung organoid cultures to determine the effect of CD47 deficiency on alveolar development. CD47-knockout mice and endothelial- specific TSP1-knockout mice will be used to test the role of TSP1-CD47 interactions in the response to neonatal hyperoxia-mediated lung vascular injury in vivo. In Aim 2, we will define the mechanisms by which TSP1-dependent Rspo2 mediates pulmonary vascular development by evaluating ligand response in vitro and after neonatal hyperoxic injury in vivo. Recombinant Rspo2 will be used in neonatal lung organoid cultures and in assays for endothelial cell proliferation, migration and tube formation. We will determine if lung endothelial cell secreted factors, and more specifically, Rspo2, is sufficient for prevention of neonatal hyperoxia-mediated lung injury. These studies will elucidate mechanisms by which lung endothelial cells drive critical outcomes in development. This work will determine if defective TSP1-dependent signaling is a mechanism of interrupted lung development in premature infants with BPD, leading to new ways to direct therapy for BPD.

总结 内皮细胞在肺发育和体内平衡的多个方面发挥关键作用，然而， 调节血管发生的精确机制，以及内皮细胞如何向其他细胞类型发出信号， 肺发育的协调性还没有很好的定义。对这些过程的准确监管对于 确保适当的肺部发育和预防支气管肺发育不良（BPD），一种早产儿的肺部疾病， 这往往会导致终生的并发症。BPD的特征是血管受损， 肺发育我们创建了三维（3D）共培养系统，其中肺内皮细胞 （LuMEC）驱动推定的肺干细胞BASC产生细支气管或肺泡类器官。这些 类器官培养物提供了模拟内皮细胞之间发生的相互作用的机会， 肺泡细胞在肺的发育过程中。利用这个系统，我们发现了一个新的BMP 4-NFATc 1- 成人肺内皮细胞和上皮细胞中的血小板反应蛋白（TSP 1）通路。我们发现TSP 1， 称为抗血管生成因子，是肺泡类器官发育所必需的。我们还确定 R-spondin 2（Rspo 2），其在其他系统中调节Wnt信号传导，作为BMP 4调节的蛋白，其可能是 内皮细胞中TSP 1的下游。TSP 1调节肺血管平滑肌细胞增殖的机制 发育和肺泡化是未知的。我们假设内皮源性TSP 1是由 BMP 4-NFATc 1信号传导抑制肺血管发育，并且是适当的肺发育所必需的。 此外，我们认为血管性肺损伤，如高氧暴露，损害TSP 1信号， 从而改变血管生成并阻断肺泡发育。在目标1中，我们将确定TSP 1- CD 47相互作用调节血管发生和肺发育。小鼠肺内皮细胞缺陷 暴露于高氧将用于肺类器官共培养物中，以测试受损内皮细胞 肺泡化的信号。类似地，来自基因敲除小鼠的细胞将用于新生儿肺类器官培养物中。 以确定CD 47缺乏对肺泡发育的影响。CD 47基因敲除小鼠和内皮细胞 将使用特定的TSP 1敲除小鼠来测试TSP 1-CD 47相互作用在对以下的应答中的作用： 新生儿体内高氧介导的肺血管损伤。在目标2中，我们将定义 TSP 1依赖性Rspo 2通过评估体外配体反应介导肺血管发育， 新生儿体内高氧损伤后。重组Rspo 2将用于新生儿肺类器官培养物， 在内皮细胞增殖、迁移和管形成的测定中。我们将确定肺内皮细胞 细胞分泌因子，更具体地说，Rspo 2，足以预防新生儿高氧介导的 肺损伤这些研究将阐明肺内皮细胞驱动关键结果的机制， 发展这项工作将确定是否有缺陷的TSP 1依赖性信号传导是一种中断的机制， 肺发育的早产儿与BPD，导致新的方法来指导治疗BPD。