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Pericytes and postnatal alveolarization: Role of hypoxia inducible factors

周细胞和出生后肺泡化：缺氧诱导因素的作用

基本信息

批准号：
10467727
负责人：
Cristina Maria Alvira
金额：
$ 64.65万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10467727
关键词：
Address Affect Alveolar Area Basement membrane Biological Birth Blood Vessels Blood capillaries Brain Bronchopulmonary Dysplasia Caliber Cell Communication Cell Proliferation Cells ChIP-seq Chronic lung disease Coculture Techniques Contracts Data Development Disease Endothelial Cells Enterobacteria phage P1 Cre recombinase Erythropoietin Exposure to Gases Gene Expression Profile Genes Genetic Goals Growth Hyperoxia Hypoxia Hypoxia Inducible Factor Image Impairment In Vitro Individual Injury Knockout Mice Life Lung Lung Capacity Lung diseases Mechanical ventilation Mediating Mesenchymal Microcirculation Molecular Molecular Computations Morphogenesis Mus Myofibroblast Natural regeneration Neonatal Oxygen Pathway interactions Pattern Pericytes Phenotype Physiological Play Polysaccharides Pregnancy Premature Infant Proliferating Regulation Reporter Resolution Role Signal Transduction Smooth Muscle Myocytes Stimulus Surface Testing Time Vascular Endothelial Growth Factors Vascular Smooth Muscle angiogenesis carbohydrate binding protein cell motility computerized tools experimental study in vivo infancy insight loss of function lung development migration mouse model neonatal exposure neonatal mice normoxia paracrine postnatal postnatal development preservation prevent promoter pup sensor single-cell RNA sequencing therapy development transcription factor transcriptome transcriptomics

项目摘要

During alveolarization, the final stage of lung development, angiogenesis drives the exponential, postnatal increase in gas-exchange surface area. Pericytes play a dual role in angiogenesis. Initially, pericytes stimulate and guide endothelial cells (EC) during early microvascular growth and subsequently constrain EC proliferation and migration during vessel stabilization. However, the molecular mechanisms that regulate pericyte phenotype to drive these distinct roles in vascular growth and stability remain poorly defined. Addressing this gap may motivate the development of therapies to treat neonatal lung diseases marked by compromised angiogenesis, including bronchopulmonary dysplasia. O2-sensitive transcription factors, termed hypoxia-inducible factors (HIF) are central regulators of angiogenesis. Our preliminary data suggest that HIF activity in select subsets of lung mesenchymal cells (MC) is required for postnatal angiogenesis and alveolarization. Genetic gain- and loss-of- function studies using Tagln promoter driven Cre-recombinase (a gene expressed by multiple MC including pericytes), demonstrated that Tagln-specific HIF stabilization preserved pulmonary vascular and alveolar growth in hyperoxia. Conversely, Tagln-specific Hif-1a deletion impaired pulmonary angiogenesis and alveolarization even in normoxia. However, the specific Tagln-expressing MC responsible was not identified. Our single cell transcriptomic studies point to a unique, hyperoxia-sensitive, developmental role for pericytes and HIF signaling in pericytes during postnatal lung development. These studies identified: (i) marked changes in the transcriptome of early (P7) versus late (P21) pericytes with blood vessel morphogenesis as the most enriched biologic pathway; (ii) a peak in both proliferating pericytes and microvascular EC at P7; (iii) persistent Hif-1a, -2a, and HIF downstream target expression in pericytes, including Rgs5, a gene that marks activated, angiogenic pericytes, and Lgals1, a gene encoding a secreted, pro-angiogenic, carbohydrate-binding protein; and (iv) hyperoxia-induced loss of ~90% of pericytes, all proliferating pericytes, and suppressed HIF-dependent, pericyte-EC interactions. These observations suggest the overall working hypothesis that HIF-mediated alterations in pericyte phenotype during postnatal development modulate the pulmonary angiogenesis that drives alveolarization. that will be tested in 3 specific aims. Aim 1 will use genetic mouse models, primary pericyte cultures, and ChIP-Seq to define the developmental role of HIF in pericytes on lung vascular growth. Aim 2 will use primary pericyte and EC co-cultures and loss of function strategies to determine if developmental regulation of select HIF-regulated targets in lung pericytes modulate pericyte phenotype and EC angiogenic function. Aim 3 will use HIF reporter mice, deep scRNA-Seq and genetic lineage tracing to determine if hyperoxia suppresses pericyte HIF-signaling, and disrupts pericytes fate and ontogeny during late lung development. By focusing on the lung pericyte, at single cell resolution, these studies will provide insight into pulmonary vascular development and identify strategies to promote lung growth and regeneration.

在肺泡化（肺发育的最后阶段）期间，血管生成驱动指数级的出生后气体交换表面积增加。周细胞在血管生成中发挥双重作用。最初，周细胞刺激并在早期微血管生长过程中引导内皮细胞 (EC)，随后抑制 EC 增殖和船舶稳定期间的迁移。然而，调节周细胞表型的分子机制驱动血管生长和稳定性的这些独特作用仍然不明确。解决这一差距可能会促进治疗以血管生成受损为特征的新生儿肺部疾病的治疗方法的开发，包括支气管肺发育不良。 O2 敏感转录因子，称为缺氧诱导因子 (HIF) 是血管生成的中央调节因子。我们的初步数据表明，选定的肺亚群中的 HIF 活性间充质细胞 (MC) 是出生后血管生成和肺泡化所必需的。遗传获得和丧失使用 Tagln 启动子驱动的 Cre 重组酶（由多个 MC 表达的基因，包括周细胞），证明 Tagln 特异性 HIF 稳定可以保护肺血管和肺泡在高氧条件下生长。相反，Tagln 特异性 Hif-1a 缺失会损害肺血管生成，即使在含氧量正常的情况下也会出现肺泡化。然而，具体表达 Tagln 的 MC 尚未确定。我们的单细胞转录组研究指出周细胞和细胞具有独特的、高氧敏感的发育作用出生后肺部发育过程中周细胞中的 HIF 信号传导。这些研究发现：(i) 显着变化在早期 (P7) 与晚期 (P21) 周细胞的转录组中，血管形态发生最为明显丰富的生物途径； (ii) 增殖周细胞和微血管 EC 在 P7 处达到峰值； (三) 坚持不懈 Hif-1a、-2a 和 HIF 下游目标在周细胞中表达，包括 Rgs5，一种标记激活的基因，血管生成周细胞和 Lgals1，一种编码分泌性促血管生成碳水化合物结合蛋白的基因； (iv) 高氧诱导约 90% 的周细胞损失，所有增殖的周细胞，并抑制 HIF 依赖性，周细胞-EC 相互作用。这些观察结果表明 HIF 介导的总体工作假设出生后发育过程中周细胞表型的改变调节肺血管生成驱动肺泡化。这将在 3 个具体目标中进行测试。目标 1 将使用基因小鼠模型，主要周细胞培养物和 ChIP-Seq 来定义周细胞中 HIF 对肺血管生长的发育作用。目标 2 将使用原代周细胞和 EC 共培养以及功能丧失策略来确定发育是否肺周细胞中选定 HIF 调节靶点的调节调节周细胞表型和 EC 血管生成功能。目标 3 将使用 HIF 报告小鼠、深度 scRNA 测序和遗传谱系追踪来确定是否存在高氧血症抑制周细胞 HIF 信号传导，并在肺部发育后期破坏周细胞的命运和个体发育。经过这些研究以单细胞分辨率关注肺周细胞，将提供对肺血管的深入了解开发并确定促进肺部生长和再生的策略。