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

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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.