Pericytes and postnatal alveolarization: Role of hypoxia inducible factors

在肺泡化，肺发育的最后阶段，血管生成驱动指数，出生后 气体交换表面积增加。周细胞在血管生成中起着双重作用。最初，周细胞刺激 并在早期微血管生长期间引导内皮细胞(EC)，随后抑制EC的增殖 以及在船只稳定期间的迁移。然而，调控周细胞表型的分子机制 对于这些在血管生长和稳定性中的不同作用，目前还没有明确的定义。解决这一差距可能会 推动开发治疗以血管生成受损为特征的新生儿肺部疾病的疗法， 包括支气管肺发育不良。氧敏感转录因子，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，在选定的肺组织亚群中，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细胞。遗传得失-- Tagln启动子驱动的Cre重组酶(一种多MC表达的基因，包括 周细胞)，证明了Tagln特异性的HIF稳定保存了肺血管和肺泡 在高氧环境中生长。相反，Tagln特异性Hif-1a缺失会损害肺血管生成和 肺泡化即使在常氧状态下也是如此。然而，具体表达Tagln的MC尚未确定。我们的 单细胞转录学研究指出周细胞和 出生后肺发育中周细胞中的低氧诱导因子信号。这些研究确定：(I)显著变化 在早期(P7)和晚期(P21)周细胞的转录组中，血管形态发生最多 丰富的生物途径；(Ii)增殖周细胞和微血管内皮细胞在P7出现高峰；(Iii)持续 HIF-1a、-2a和HIF下游靶基因在周细胞中的表达，包括RGS5，一个标记激活的基因， 血管生成周细胞，以及Lgals1，一种编码分泌的、促血管生成的碳水化合物结合蛋白的基因； 以及(Iv)高氧导致约90%的周细胞丧失，所有增殖的周细胞，并抑制HIF依赖， 周细胞与内皮细胞的相互作用。这些观察结果表明，HIF介导的总体工作假说 出生后发育过程中周细胞表型的变化对肺血管生成的调节作用 这推动了牙槽的形成。这将在3个具体目标上进行测试。AIM 1将使用遗传小鼠模型，主要 周细胞培养和CHIP-SEQ以确定周细胞中HIF在肺血管生长中的发育作用。 目标2将使用原代周细胞和内皮细胞共培养和功能丧失策略来确定是否发育 肺周细胞HIF调控靶点调控周细胞表型和EC血管生成 功能。AIM 3将使用HIF报告小鼠、深层scRNA-Seq和遗传谱系追踪来确定高氧血症 抑制周细胞缺氧诱导因子信号，扰乱肺发育后期周细胞的命运和个体发育。通过 以肺周细胞为研究对象，在单细胞分辨率下，这些研究将提供对肺血管的洞察。 发展和确定促进肺生长和再生的战略。

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.