Origin and maturation of the pulmonary lymphatic endothelium
Norman Jr., Timothy Alfred
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The lymphatic vasculature is composed of lymphatic endothelial cells (LECs) that coalesce into a branched hierarchy of small capillaries and larger collecting vessels that regulate interstitial fluids, lipid uptake and immunity. Few studies have focused on pulmonary lymphatic system. To fill these critical knowledge gaps, we interrogated the fetal maturation program of lymphatic endothelium, and we provide evidence that CSF1R-lineage progenitors contribute to LECs in the lung during a temporally defined period in early postnatal life. The pulmonary lymphatic system is required for fluid clearance and air breathing at birth, suggesting a prenatal maturation program. To interrogate this, we developed a cell sorting strategy to enrich pulmonary LECs by their unique cell surface immunophenotype (CD45-, EPCAM-, CD31+, VEGFR3+, PDPN+, LYVE1+) for transcriptional profiling. These experiments highlighted the coordinate down-regulation of genes involved in “cell cycle”, and “mRNA processing” along with coordinate upregulation of “complement/coagulation cascade”, “lipid metabolism”, and “angiogenesis” genes from embryonic day E16.5 to E18.5. The most significantly enriched gene set corresponded to the “interferon-alpha/beta signaling” pathway which was confirmed with qRT-PCR and in-situ hybridization. These data provide the first description of the transcriptional landscape of fetal pulmonary LEC maturation. During development, all LECs are thought to originate from embryonic veins, however multiple studies have suggested a myeloid origin for a subset of LECs. A relationship between myeloid cells and the pulmonary LECs has not been elucidated. Here, we used myeloid-specific inducible CSF1R-CreERtdTomato lineage tracing mice and identified rare, single cells that co-expressed CSF1R- CreERtdTomato and Prox1, the master lymphatic regulator, in the postnatal day 3 lung. This process was temporally restricted to the early postnatal period. Lineage tracing with additional myeloid-Cre mice (CSF1R-iCre and CX3CR1-Cre) also showed contribution to postnatal LECs. To determine the biological significance of CSF1R-derived LECs to postnatal lung biology, we performed conditional Prox1 loss of function experiments. CSF1R-CreER mediated deletion of Prox1 resulted in lymphatic hypoplasia, edematous foci and clotting. These findings suggest that early postnatal CSF1R+ progenitors contribute to the pulmonary lymphatic endothelium and that vascular clotting may result from lymphatic malformation/dysfunction.
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