CXCL12 signaling axis in pulmonary arterial heterogeneity, development, and disease

项目摘要 发展范式的重新参与经常指导疾病的发病机制。虽然关键在于 生长和分化的组织形成，这些途径的异常表达导致显着的 病理学如癌症、自身免疫和慢性疾病。同样，正常的胎儿程序中断， 在发育过程中，肺可导致显著的肺血管发育不良， 成人，这表明在预防疾病的共同作用。因此，了解细胞和分子 协调肺血管发展的机制将有助于确定肺部疾病的治疗靶点 为了研究驱动发育的细胞和分子途径，我们将重点放在动脉上， 内皮细胞是调节血管阻力和预防肺损伤的重要血管区室。 肺动脉高压（PH）等疾病。我们使用Cxcl 12DsRed标记动脉内皮细胞（EC）， 荧光报告小鼠，其中在表达标志性动脉的细胞中产生红色荧光蛋白 基因，Cxcl12。我们分离Cxcl12+细胞并进行单细胞RNA测序，以评估细胞内的 肺内皮细胞内的异质性。结合细胞注释、基因本体分析和空间 转录组学揭示了发育中动脉内皮细胞的空间和功能不同的新亚群， 肺。此外，我们还发现了从动脉到毛细血管的CXCL 12形态发生梯度，这表明CXCL 12是一种重要的细胞因子。 对肺动脉生长的趋触机制。此外，形态发生梯度的破坏导致 在肺血管发育不全和异常分支中，我们通过应用独特的方法进行定量， 肺。 在这项提案中，我们正在评估CXCL12在发育和疾病中的扩大作用。空间 Cxcl12及其受体Cxcr4和Ackr3的转录组学促进了我们的假设，即动脉生长 并且组装受CXCL 12形态发生梯度控制，其引导毛细血管的细胞迁移。 将祖细胞转移到远端动脉。此外，这种表达模式被观察到成年，我们 假设肺动脉树包含空间和功能不同的亚群 在整个生命周期中保存。我们将使用遗传小鼠模型和单克隆抗体来测试这些假设。 细胞技术揭示动脉异质性和CXCL12协调的肺血管机制 发展 目标1。明确CXCL12信号在肺血管发育中的时空作用。 目标二。评估发育性肺病中CXCL12依赖性EC异质性和信号传导。 目标3。识别和表征进化保守的近端和远端动脉EC。

PROJECT SUMMARY Reengagement of developmental paradigms frequently instructs disease pathogenesis. While critical for growth and differentiation for tissue formation, aberrant expression of these pathways leads to significant pathology such as cancer, autoimmunity, and chronic disease. Similarly, disruption of normal fetal programs in the lung can result in significant pulmonary vascular hypoplasia during development and loss of homeostasis in the adult, suggesting a common role in prevention of disease. Thus, understanding the cellular and molecular mechanisms orchestrating pulmonary vascular development will help identify therapeutic targets for lung disease To investigate the cellular and molecular pathways driving development, we focused on the arterial endothelium, a vascular compartment important in the regulation of vascular resistance and prevention of lung diseases such pulmonary hypertension (PH). We labeled arterial endothelial cells (ECs) using the Cxcl12DsRed fluorescent reporter mouse in which a red fluorescent protein is produced in cells expressing the hallmark arterial gene, Cxcl12. We isolated Cxcl12+ cells and performed single cell RNA sequencing to assess cellular heterogeneity within the pulmonary endothelium. Combined cell annotation, gene ontology analysis, and spatial transcriptomics revealed spatially and functionally distinct novel subpopulations of arterial ECs in the developing lung. In addition, we discovered a CXCL12 morphogen gradient from arteries to capillaries, suggesting a haptotaxis mechanism to pulmonary artery growth. Furthermore, disruption of the morphogen gradient resulted in pulmonary vascular hypoplasia and aberrant branching that we quantified by applying unique methods for the lung. In this proposal, we are assessing an expanded role for CXCL12 in development and disease. Spatial transcriptomics for Cxcl12 and its receptors, Cxcr4 and Ackr3, promote our hypothesis that arterial growth and assembly is governed by a CXCL12 morphogen gradient directing cell migration of capillary progenitor cells to the distal artery. In addition, this pattern of expression is observed into adulthood, and we hypothesize that the pulmonary arterial tree contains spatially and functionally distinct subpopulations conserved throughout the lifespan. We will test these hypotheses using genetic mouse models and single cell technology to uncover mechanisms of arterial heterogeneity and CXCL12-orchestrated pulmonary vascular development. Aim 1. To define the spatiotemporal role of CXCL12 signaling in pulmonary vascular development. Aim 2. To assess CXCL12-dependent EC heterogeneity and signaling in developmental lung disease. Aim 3. To identify and characterize evolutionarily conserved proximal and distal arterial ECs.