Developmental diversity and unique sensitivity to injury of lung endothelial subtypes during a period of rapid postnatal growth

Abstract

BackgroundEndothelial cells (EC) sit at the forefront of dramatic physiologic changes occurring in the pulmonary circulation during late embryonic and early postnatal life. First, as the lung moves from the hypoxic fetal environment to oxygen-rich postnatal environment, marked changes in pulmonary EC structure and function facilitate a marked increase in blood flow from the placenta to the lungs. Subsequently, pulmonary angiogenesis expands the microvasculature to drive exponential distal lung growth during early postnatal life. Yet, how these marked physiologic changes alter distinct EC subtypes to facilitate the transition of the pulmonary circulation and regulate vascular growth and remodeling remains incompletely understood.MethodsIn this report, we employed single cell RNA-transcriptomics and in situ RNA imaging to profile pulmonary EC in the developing mouse lung from just before birth through this period of rapid postnatal growth.ResultsMultiple, transcriptionally distinct macro- and microvascular EC were identified in the late embryonic and early postnatal lung, with gene expression profiles distinct from their adult EC counterparts. A novel arterial subtype, unique to the developing lung localized to the distal parenchyma and expressed genes that regulate vascular growth and patterning. Birth particularly heightened microvascular diversity, inducing dramatic shifts in the transcriptome of distinct microvascular subtypes in pathways related to proliferation, migration and antigen presentation. Two distinct waves of EC proliferation were identified, including one just prior to birth, and a second during early alveolarization, a time of exponential pulmonary angiogenesis. Chronic hyperoxia, an injury that impairs parenchymal and vascular growth, induced a common gene signature among all pulmonary EC, unique alterations to distinct microvascular EC subtypes, and disrupted EC-EC and EC-immune cell cross talk.ConclusionsTaken together, these data reveal tremendous diversity of pulmonary EC during a critical window of postnatal vascular growth, and provide a detailed molecular map that can be used to inform both normal vascular development and alterations in EC diversity upon injury. These data have important implications for lung diseases marked by dysregulated angiogenesis and pathologic pulmonary vascular remodeling.

Lung mesenchymal cell diversity rapidly increases at birth and is profoundly altered by hyperoxia

“…In these experiments, we identified a previously undescribed cluster of fibroblasts (MHF), present only in male hyperoxia exposed mice ( Figures 7G-I ). No novel cell types, including sex-specific ones, were observed in the immune [13] or endothelial [20] compartments in our previous studies. The spatial distribution of MHF, superimposed upon expression of cell contractility genes, support the proposition that the cells possess meaningful pathophysiologic significance.…”

“…The dramatic temporal shifts in the transcriptome of fibroblasts and ASM/MyoF resemble those seen in Mac I-II-III macrophages [13] and Car4-capillary endothelial cells [20] . In contrast, phenotypic shifts in mural cells were gradual but progressive, without marked changes between time points, reminiscent of alterations in multiple immune subtypes including monocytes (Mac V) [13] and in Car4 + capillaries [20,57] , macrovascular cells, and lymphatic cells within the endothelium [20] . These findings indicate that select cell types perform a similar biological function throughout this developmental window, while others need to adapt transcriptionally to the distinct needs of each stage of tissue growth.…”

“…Lung development continues after birth with a rapid increase in the number of alveoli [10] . While insight into the contributions of the cellular constituents of the lung across development is increasing [13,20] , definitive information surrounding the composition, interactions and functions of lung mesenchymal cells remains elusive. In particular, there is still great ambiguity in the definition and naming of cell subtypes across the lung mesenchyme, especially during development [27,28,39] .…”

“…We thereby distinguished three coarse mesenchymal cell types: fibroblasts marked by either Col13a1 or C ol14a1 [16,17] , airway smooth muscle cells or myofibroblasts (ASM/MyoF) marked by Tgfbi [18] , and mural cells marked by Pdgfrb [19] ( Figure 1C ). No cluster demonstrated high expression of both Tgfbi and Pdgfrb , previously described as vascular smooth muscle cells (VSM) markers [20] , arguing for the importance of clarifying VSM annotations. Cell type abundance formed an interesting temporal pattern: fibroblasts were the majority cell type at all time points except P7, ASM/MyoF peaked at P7, and the relative abundance of mural cells gradually increased with maturation ( Figure 1D ).…”

“…Other cell type short names: EC = endothelial cell, NP emb EC = nonproliferative embryonic EC, AT1/2 = epithelial alveolar type 1/2. Data sources: endothelial cells [20] , epithelial cells [39] , and immune cells [13] .…”