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Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

ABSTRACT: Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the adipocyte-specific promoter adiponectin (ADN), to determine how chylothorax forms. Surprisingly, 100% of adult ADN-VEGF-C mice developed chylothorax within 7 days. Rapid, consistent appearance of chylothorax enabled us to examine the step-by-step development in otherwise normal adult mice. Dynamic imaging with a fluorescent tracer revealed that lymph in the thoracic duct of these mice could enter the thoracic cavity by retrograde flow into enlarged paravertebral lymphatics and subpleural lymphatic plexuses that had incompetent lymphatic valves. Pleural mesothelium overlying the lymphatic plexuses underwent exfoliation that increased during doxycycline exposure. Together, the findings indicate that chylothorax in ADN-VEGF-C mice results from retrograde flow of chyle from the thoracic duct into lymphatic tributaries with defective valves. Chyle extravasates from these plexuses and enters the thoracic cavity through exfoliated regions of the pleural mesothelium.

SUBMITTER: Nitschke M

PROVIDER: S-EPMC5808174 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

Nitschké Maximilian M Bell Alexander A Karaman Sinem S Amouzgar Meelad M Rutkowski Joseph M JM Scherer Philipp E PE Alitalo Kari K McDonald Donald M DM

The American journal of pathology 20170703 9

Chylous pleural effusion (chylothorax) frequently accompanies lymphatic vessel malformations and other conditions with lymphatic defects. Although retrograde flow of chyle from the thoracic duct is considered a potential mechanism underlying chylothorax in patients and mouse models, the path chyle takes to reach the thoracic cavity is unclear. Herein, we use a novel transgenic mouse model, where doxycycline-induced overexpression of vascular endothelial growth factor (VEGF)-C was driven by the a ...[more]

PMID: 28683257

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Project description:ObjectiveInflammatory-erosive arthritis is exacerbated by dysfunction of joint-draining popliteal lymphatic vessels (PLVs). Synovial mast cells are known to be pro-inflammatory in rheumatoid arthritis (RA). In other settings they have anti-inflammatory and tissue reparative effects. Herein, we elucidate the role of mast cells on PLV function and inflammatory-erosive arthritis in tumor necrosis factor transgenic (TNF-tg) mice that exhibit defects in PLVs commensurate with disease progression.MethodsWhole mount immunofluorescent microscopy, toluidine blue stained histology, scanning electron microscopy, and in silico bioinformatics were performed to phenotype and quantify PLV mast cells. Ankle bone volumes were assessed by μCT, while corresponding histology quantified synovitis and osteoclasts. Near-infrared indocyanine green imaging measured lymphatic clearance as an outcome of PLV draining function. Effects of genetic MC depletion were assessed via comparison of 4.5-month-old WT, TNF-tg, MC deficient KitW-sh/W-sh (cKit-/-), and TNF-tg x cKit-/- mice. Pharmacological inhibition of mast cells was assessed by treating TNF-tg mice with placebo or cromolyn sodium (3.15mg/kg/day) for 3-weeks.ResultsPLVs are surrounded by MCT+/MCPT1+/MCPT4+ mast cells whose numbers are increased 2.8-fold in TNF-tg mice. The percentage of peri-vascular degranulating mast cells was inversely correlated with ICG clearance. A population of MCT+/MCPT1-/MCPT4- mast cells were embedded within the PLV structure. In silico single-cell RNA-seq (scRNAseq) analyses identified a population of PLV-associated mast cells (marker genes: Mcpt4, Cma1, Cpa3, Tpsb2, Kit, Fcer1a & Gata2) with enhanced TGFβ-related signaling that are phenotypically distinct from known MC subsets in the Mouse Cell Atlas. cKit-/- mice have greater lymphatic defects than TNF-tg mice with exacerbation of lymphatic dysfunction and inflammatory-erosive arthritis in TNF-tg x cKit-/- vs. TNF-Tg mice. Cromolyn sodium therapy stabilized PLV mast cells, increased TNF-induced bone loss, synovitis, and osteoclasts, and decreased ICG clearance.ConclusionsMast cells are required for normal lymphatic function. Genetic ablation and pharmacological inhibition of mast cells exacerbates TNF-induced inflammatory-erosive arthritis with decreased lymphatic clearance. Together, these findings support an inflammatory role of activated/degranulated peri-PLV mast cells during arthritic progression, and a homeostatic role of intra-PLV mast cells, in which loss of the latter dominantly exacerbates arthritis secondary to defects in joint-draining lymphatics, warranting investigation into specific cellular mechanisms.

Project description:Lymphatic malformations (LM) are characterized by the overgrowth of lymphatic vessels during pre- and postnatal development. Macrocystic, microcystic and combined forms of LM are known. The cysts are lined by lymphatic endothelial cells (LECs). Resection and sclerotherapy are the most common treatment methods. Recent studies performed on LM specimens in the United States of America have identified activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) gene in LM. However, whole tissue but not isolated cell types were studied. Here, we studied LM tissues resected at the University Hospitals Freiburg and Regensburg, Germany. We isolated LECs and fibroblasts separately, and sequenced the commonly affected exons 8, 10, and 21 of the PIK3CA gene. We confirm typical monoallelic mutations in 4 out of 6 LM-derived LEC lines, and describe two new mutations i.) in exon 10 (c.1636C>A; p.Gln546Lys), and ii.) a 3bp in-frame deletion of GAA (Glu109del). LM-derived fibroblasts did not possess such mutations, showing cell-type specificity of the gene defect. High activity of the PIK3CA-AKT- mTOR pathway was demonstrated by hyperphosphorylation of AKT-Ser473 in all LM-derived LECs (including the ones with newly identified mutations), as compared to normal LECs. Additionally, hyperphosphorylation of ERK was seen in all LM-derived LECs, except for the one with Glu109del. In vitro, the small molecule kinase inhibitors Buparlisib/BKM-120, Wortmannin, and Ly294002, (all inhibitors of PIK3CA), CAL-101 (inhibitor of PIK3CD), MK-2206 (AKT inhibitor), Sorafenib (multiple kinases inhibitor), and rapamycin (mTOR inhibitor) significantly blocked proliferation of LM-derived LECs in a concentration-dependent manner, but also blocked proliferation of normal LECs. However, MK-2206 appeared to be more specific for mutated LECs, except in case of Glu109 deletion. In sum, children that are, or will be, treated with kinase inhibitors must be monitored closely.

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Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

Publications

Retrograde Lymph Flow Leads to Chylothorax in Transgenic Mice with Lymphatic Malformations.

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