The emerging importance of lymphatics in health and disease: an NIH workshop report

The lymphatic system (LS) is composed of lymphoid organs and a network of vessels that transport interstitial fluid, antigens, lipids, cholesterol, immune cells, and other materials in the body. Abnormal development or malfunction of the LS has been shown to play a key role in the pathophysiology of many disease states. Thus, improved understanding of the anatomical and molecular characteristics of the LS may provide approaches for disease prevention or treatment. Recent advances harnessing single-cell technologies, clinical imaging, discovery of biomarkers, and computational tools have led to the development of strategies to study the LS. This Review summarizes the outcomes of the NIH workshop entitled “Yet to be Charted: Lymphatic System in Health and Disease,” held in September 2022, with emphasis on major areas for advancement. International experts showcased the current state of knowledge regarding the LS and highlighted remaining challenges and opportunities to advance the field.


Date
Events and discoveries that advanced lymphatic research 1938 Henri Rouvière publishes textbook "Anatomy of the Human Lymphatic System", still regarded as the most comprehensive treatise on lymphatic anatomy (35).
1939 Paul Patek performs a detailed assessment of the morphology and anatomy of cardiac lymphatics in a wide range of mammals (36) 1940s Use of radioactive iodine to demonstrate that lymph originates from the blood stream (16). 1949 First documented study in PubMed describing the presence of intrinsic contractility in human lymphatics (37).Original description of pumping activity of lymphatic vessels described by Arnold Heller in 1869 (38). 1950s Lymphoscintigraphy is first established in the 1950s and is still regarded as the "gold-standard" for lymphedema diagnosis (39).
1952 Kinmonth achieves the first good lymphangiogram through injection of diodone, a water-soluble radiopaque contrast medium, in humans (40).

1958-1965
Leonetto Comparini and colleagues make several significant contributions related to the molecular and morphological characterization of lymphatic capillaries, pre-collecting lymphatic vessels, and collecting lymphatic vessels.He additionally contributed reconstructions of the hepatic lymphatic system (3,41).Ernest Gould is the first to use the term "sentinel node" in his pathology report of "lymph node with metastatic tumor" (42).
2015 Workshop: The Third Circulation: Lymphatics as Regulators in Health and Disease (83).

2016
The Human Cell Atlas, an international effort to characterize every cell in the human body at single cell resolution using advanced technologies, is established (84).

2018
Major trans-NIH effort, the Human BioMolecular Atlas Program (HuBMAP), supported by the NIH Common Fund, brings together biologists, pathologists, and data scientists to create an open and global platform to map the cells of all the major human organ systems, including the lymphatic system (85,86).

2018
The clinical application of intranodal mesenteric lymphangiography for managing postoperative recalcitrant chylous ascites is evaluated in a feasibility study (87).

2021
Workshop: Yet to Be Charted: Mapping the Lymphatic System Across Body Scales and Expertise Domains (88).

2021
Activating somatic mutation in KRAS was identified in a patient with Gorham-Stout disease (GSD).A GSD mouse model was developed revealing lymphatic developmental defects with significantly fewer lymphatic valves (89).2022 Workshop: Yet to Be Charted: Lymphatic System in Health and Disease (90).

2023-Present
Ongoing efforts to establish a National Commission on Lymphatic Diseases after Congressional Mandate on FY2021 appropriations for the Department of Health and Human Services (92).

Supplemental Table 1. A comprehensive timeline of lymphatic discoveries and NIH-led activities and funding opportunities.
The field of lymphatic research spans several millennia, reflecting a timeless desire to understand the system and its parts.Summary of landmark findings, international workshops, and NIH-led activities is provided with key references for further review.For a more extensive account of the scholars, discoveries, and priority disputes we refer the reader to several excellent reviews on this topic (3-5, 7, 8, 11, 16, 20).

LYVE1 Lyve1CreERT2
Inducible Cre recombinase-estrogen receptor construct targeting lymphatic epithelial cells (LECs).This transgenic mouse allows for visualization and selective deletion of LECspecific genes in entire lymphatic vessels or LEC progenitors depending on the dosing schedule of 4-hydroxytamoxifen and genetic cross to floxed mice (98).

Podoplanin Pdpn-GFPCre
Transgenic mouse expressing Cre under control of a 5' upstream regulatory region of the Pdpn gene, providing a better tool to visualize lymphatics and their function in mice (99).

Prox1 prox1-mOrange2-pA-BAC
Prox1 promoter driven expression of fluorescent protein mOrange2 for intravital visualization of lymphatic growth and function in fetal and adult mice (101).

Prox1 Prox1-CreERT2
Transgenic line expressing tamoxifen-inducible Cre recombinase (CreER T2 ) under control of the Prox1 gene promoter.When crossed to transgenic mice carrying fluorescent reporter proteins or floxed genes, the resulting progeny can be used to visualize lymphatics and/or perform functional studies on conditional knockouts, respectively (102).
Prox1 ProxTom Contains all Prox1 regulatory sequences for expression of RFP tdTomato in lymphatic vessels.Demonstrates intense expression in endogenous lymphatic vessels (104).

Prox1
Prox1-Cre-tdTomato tdTomato reporter mouse crossed with line expressing Cre recombinase under control of the Prox1 promoter (Prox1-Cre-ERT2 line (102) allows for intravital microscopy of dendritic cell migration into and within lymphatic vessels and fluorescenceactivated single cell analysis of lymphatic endothelial cells (105).

Prox1
Prox1-GFP/Flk1::myr-mCherry mice Dual reporter system where lymphatic vessels emit green and blood vessels emit red fluorescence, allowing for simultaneous observation of lymphangiogenesis and angiogenesis in vivo (106).

Prox1
Prox1-GFP Prox1-GFP mice on the wild-type C57BL/6 background.Applications include intravital imaging of newly formed lymphatic vessels and valves in the cornea to observe lymphangiogenesis stimulated by injury and/or therapeutic intervention (107).

Mouse strain Description and/or application(s) Year
Prox1 Prox1-EGFP BAC Transgenic lymphatic reporter rat expressing EGFP via BAC with mouse Prox1 regulatory elements.Allows for visualization of all lymphatic vessels including the lymphatics of the central nervous system and Schlemm's canal (109).
Use of tamoxifen-inducible LEC-specific Cre line (Prox1Cre ERT2) crossed to Foxo1 flox/flox mice to evaluate the role of Foxo1 on lymphatic valve function in a temporally controlled manner.Deletion of Foxo1 in these mice activated the lymphatic valve formation process.

VEGFC VEGFC/LacZ
LacZ reporter gene placed in exon of Vegfc to create postnatal lethal Vegfc knockout mouse.VEGFC was shown to be dispensable for LEC lineage commitment but critical for sprouting proceeding lymphatic endothelial specification in embryonic mice (60).

VEGFR2
Lyve-1 (wt/Cre) ;Vegfr2 (flox/flox) Mice develop functional lymphatic vasculature in the absence of VEGFR2 but exhibit lymphatic hypoplasia (112).Genetically engineered mice expressing fluorescent reporter proteins or Cre recombinase under control of LEC-specific promoters such as Lyve1, Prox1, Vegfr3, and Pdpn have been widely used to study the lymphatic system.Here, we summarize of few of these strains and provide original references for further review.A limitation of these models is the expression of LECspecific markers by other cell types and tissues, e.g., Lyve1 by macrophages, Prox1 by cardiomyocytes, and Pdpn (Podoplanin) in the injured heart (118)(119)(120).Additional genetic strains can be found using the Mouse Genome Database (MGD; http://www.informatics.jax.org)(121).