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Hepatic lymphatic vascular system in health and disease

  • Author Footnotes
    † Equal contribution
    Jain Jeong
    Footnotes
    † Equal contribution
    Affiliations
    Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA
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  • Author Footnotes
    † Equal contribution
    Masatake Tanaka
    Footnotes
    † Equal contribution
    Affiliations
    Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
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  • Yasuko Iwakiri
    Correspondence
    Corresponding author. Address: Department of Internal Medicine, Section of Digestive Diseases, Yale School of Medicine, 1080LMP, 333 Cedar Street, New Haven, CT 06520, USA; Tel.: +1-203-785-6204, fax: +1-203-785-7273.
    Affiliations
    Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA
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  • Author Footnotes
    † Equal contribution
Published:February 11, 2022DOI:https://doi.org/10.1016/j.jhep.2022.01.025

      Summary

      In recent years, significant advances have been made in the study of lymphatic vessels with the identification of their specific markers and the development of research tools that have accelerated our understanding of their role in tissue homeostasis and disease pathogenesis in many organs. Compared to other organs, the lymphatic system in the liver is understudied despite its obvious importance for hepatic physiology and pathophysiology. In this review, we describe fundamental aspects of the hepatic lymphatic system and its role in a range of liver-related pathological conditions such as portal hypertension, ascites formation, malignant tumours, liver transplantation, congenital liver diseases, non-alcoholic fatty liver disease, and hepatic encephalopathy. The article concludes with a discussion regarding the modulation of lymphangiogenesis as a potential therapeutic strategy for liver diseases.

      Keywords

      Introduction

      The liver is recognised as the largest lymph-producing organ, accounting for nearly 25–50% of lymph passing through the thoracic duct (Fig. 1).
      • Morris B.
      The hepatic and intestinal contributions to the thoracic duct lymph.
      • Cain J.C.
      • Grindlay J.H.
      • Bollman J.L.
      • Flock E.V.
      • Mann F.C.
      Lymph from liver and thoracic duct; an experimental study.
      • Mobley W.P.
      • Kintner K.
      • Witte C.L.
      • Witte M.H.
      Contribution of the liver to thoracic duct lymph flow in a motionless subject.
      The functional importance of the hepatic lymphatic system has mainly been discussed in relation to the regulation of lymph production, which is highly influenced by haemodynamic changes in the intrahepatic microcirculation. While lymphatic vessel numbers are known to increase (via lymphangiogenesis: new lymphatic vessel formation) in many liver diseases, the mechanism of lymphangiogenesis in the liver remains poorly understood.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      ,
      • Ohtani O.
      • Ohtani Y.
      Lymph circulation in the liver.
      In addition to the maintenance of fluid homeostasis, hepatic lymphatic vessels are highly involved in the immune system and lipid metabolism as they transport various immune cells, antigens and lipids to lymph nodes. Despite these roles in key processes in the liver, hepatic lymphatic vessels have been understudied.
      Figure thumbnail gr1
      Fig. 1Hepatic lymphatic system.
      Lymph is thought to flow into lymphatic vessels located in 3 regions: portal, hepatic venous and sub-capsular areas. The illustration shows lymphatic vessels in the portal tract, the primary site of hepatic lymph drainage covering around 80% of the lymph produced by the liver. Because the hepatic sinusoids are highly permeable due to fenestrae, fluid in the hepatic sinusoids can flow through the channels traversing the limiting plate to the interstitial space of the portal tract. Endothelial cells of lymphatic capillaries have discontinuous, “button-like” junctions, which enable the efficient entry of fluid, antigens and immune cells into lymphatic capillaries. Lymphatic capillaries in the portal tract coalesce into collecting lymphatic vessels surrounded by lymphatic muscle cells outside the liver. Lymphatic muscle cells covering collecting lymphatic vessels help to pump lymph into regional lymph nodes located in the hepatic hilum and then to the cisterna chyli located at the lower end of the thoracic duct. Lymph finally drains into the left subclavian vein via the thoracic duct and returns to the systemic blood circulation. BD, bile duct; CV, central vein; ECM, extracellular matrix protein; HA, hepatic artery; HSC, hepatic stellate cell; LV, lymphatic vessel; PV, portal vein.
      In the last decade, significant advances have been made in the study of lymphatic vessels, including the identification of specific markers and the development of research tools, accelerating our understanding of their roles in tissue homeostasis and disease pathogenesis in many organs.
      • Zheng W.
      • Aspelund A.
      • Alitalo K.
      Lymphangiogenic factors, mechanisms, and applications.
      • Tammela T.
      • Alitalo K.
      Lymphangiogenesis: molecular mechanisms and future promise.
      • Norrmen C.
      • Tammela T.
      • Petrova T.V.
      • Alitalo K.
      Biological basis of therapeutic lymphangiogenesis.
      Lymphatic vessels exhibit a variety of immunoregulatory functions by expressing a wide range of chemokines and receptors. Thus, lymphatic vessels are more than just conduits that remove lymph, immune cells and cellular products from organs and tissues.
      In this review, we aim to describe fundamental aspects of the hepatic lymphatic system and its implications for liver diseases. We will discuss: i) the fundamentals of liver lymphatics, including structure, markers, lymph and lymphatic drainage; ii) lymphatics and the immune system; iii) mediators and signals leading to hepatic lymphangiogenesis; iv) lymphatics in liver diseases and their complications, including portal hypertension, ascites, malignant tumours, liver transplantation, congenital liver diseases, non-alcoholic fatty liver disease (NAFLD), and hepatic encephalopathy; v) therapeutic potential and future directions in the study of liver lymphatics. For details of the biology of the hepatic lymphatic system, other review papers are referenced in Table S1.

      Hepatic lymphatic system

      Structure of lymphatics

      A history of studies of the hepatic lymphatic system and how hepatic lymph reaches hepatic lymphatic vessels are described in an excellent review paper by Ohtani & Ohtani.
      • Ohtani O.
      • Ohtani Y.
      Lymph circulation in the liver.
      The lymphatic vascular system consists of lymphatic capillaries (also known as initial lymphatics) and collecting lymphatic vessels. Lymphatic capillaries consist of a single layer of lymphatic endothelial cells (LECs) with no coverage of “lymphatic muscle” cells. In contrast to lymphatic capillaries, collecting lymphatic vessels are covered with lymphatic muscle cells and located downstream of lymphatic capillaries. “Lymphatic vessels” are often defined as an intermediate structure between lymphatic capillaries and lymphatic collecting vessels. In this review article, however, we use the term “lymphatic vessels” to describe both lymphatic vessels and lymphatic capillaries, because lymphatic capillaries are most common in the liver. Endothelial cells of lymphatic capillaries have discontinuous “button-like” junctions, which are strikingly different from continuous “zipper-like” junctions of collecting lymphatic vessels and blood vessels.
      • Baluk P.
      • Fuxe J.
      • Hashizume H.
      • Romano T.
      • Lashnits E.
      • Butz S.
      • et al.
      Functionally specialized junctions between endothelial cells of lymphatic vessels.
      ,
      • Dejana E.
      • Orsenigo F.
      • Molendini C.
      • Baluk P.
      • McDonald D.M.
      Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees.
      The button-like junctions are thought to allow for efficient entry of fluid, antigens and immune cells into lymphatic capillaries (Fig. 2). In pathological conditions, it is reported that lymphatic capillaries lose this “button-like” structure with a change to the less permeable “zipper-like” structure.
      • Yao L.C.
      • Baluk P.
      • Srinivasan R.S.
      • Oliver G.
      • McDonald D.M.
      Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.
      ,
      • Cifarelli V.
      • Eichmann A.
      The intestinal lymphatic system: functions and metabolic implications.
      This change of junctional structure could impair transport of fluid and substances to lymphatic capillaries, thereby decreasing their clearance from tissues.
      The liver is recognised as the largest lymph-producing organ.
      Figure thumbnail gr2
      Fig. 2Button vs. Zipper-like structures.
      The lymphatic vascular system includes lymphatic capillaries (also known as initial lymphatics) and collecting lymphatic vessels. Lymphatic capillaries consist of a single layer of lymphatic endothelial cells. Endothelial cells of lymphatic capillaries are attached by anchoring filaments to surrounding extracellular matrix proteins, which support their vessel structure. Endothelial cells of lymphatic capillaries have discontinuous, “button-like” junctions, which enable the efficient entry of fluid, antigens and immune cells into lymphatic capillaries. In contrast, collecting lymphatic vessels have “zipper-like” junctions, similar to blood vessels. In pathological conditions, lymphatic capillaries lose this “button-like” structure with a change to the less permeable “zipper-like” structure, which results in impaired transport of fluid and substances to lymphatic capillaries, thereby decreasing their clearance from tissues.

      Markers of lymphatics

      Markers of LECs include vascular endothelial growth factor receptor 3 (VEGFR3),
      • Joukov V.
      • Pajusola K.
      • Kaipainen A.
      • Chilov D.
      • Lahtinen I.
      • Kukk E.
      • et al.
      A novel vascular endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) and KDR (VEGFR-2) receptor tyrosine kinases.
      ,
      • Kukk E.
      • Lymboussaki A.
      • Taira S.
      • Kaipainen A.
      • Jeltsch M.
      • Joukov V.
      • et al.
      VEGF-C receptor binding and pattern of expression with VEGFR-3 suggests a role in lymphatic vascular development.
      lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1),
      • Banerji S.
      • Ni J.
      • Wang S.X.
      • Clasper S.
      • Su J.
      • Tammi R.
      • et al.
      LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.
      ,
      • Prevo R.
      • Banerji S.
      • Ferguson D.J.
      • Clasper S.
      • Jackson D.G.
      Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.
      prospero homeobox protein 1 (Prox1)
      • Wigle J.T.
      • Harvey N.
      • Detmar M.
      • Lagutina I.
      • Grosveld G.
      • Gunn M.D.
      • et al.
      An essential role for Prox1 in the induction of the lymphatic endothelial cell phenotype.
      and podoplanin (also known as D2-40).
      • Breiteneder-Geleff S.
      • Soleiman A.
      • Kowalski H.
      • Horvat R.
      • Amann G.
      • Kriehuber E.
      • et al.
      Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.
      However, these LEC markers are also expressed in other liver cells, which has made studies of the hepatic lymphatic system challenging.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      ,
      • Tanaka M.
      • Iwakiri Y.
      Lymphatics in the liver.
      For example, VEGFR3 and Lyve1 are expressed in liver sinusoidal endothelial cells (LSECs), while Prox1 is expressed in hepatocytes. In the normal liver, the localisation of lymphatic capillaries in the portal tract allows these markers to be used for their identification. Because lymphatic capillaries are not covered by αSMA-positive smooth muscle cells, αSMA-labelling can differentiate lymphatics from blood vessels in the portal tract. In human liver specimens, podoplanin/D2-40 has frequently been used to identify lymphatic vessels. However, identification of unique LEC proteins that are not expressed in other liver cells is indispensable for advancing our understanding of lymphatic vessels in the liver.
      The presence of LECs in the liver tends to be overlooked at least in part because they only account for a very small portion of the liver EC population in the normal liver, while cirrhosis increases the contribution of LECs to total ECs by 20-fold.
      • Su T.
      • Yang Y.
      • Lai S.
      • Jeong J.
      • Jung Y.
      • McConnell M.
      • et al.
      Single-cell transcriptomics reveals zone-specific alterations of liver sinusoidal endothelial cells in cirrhosis.
      A recent study of the liver EC population using single-cell RNA sequencing analysis identified several novel genes highly expressed in LECs, but with absent or only minimal expression in LSECs and arterial/venous ECs.
      • Su T.
      • Yang Y.
      • Lai S.
      • Jeong J.
      • Jung Y.
      • McConnell M.
      • et al.
      Single-cell transcriptomics reveals zone-specific alterations of liver sinusoidal endothelial cells in cirrhosis.
      These potential liver LEC markers include multimerin 1, Rassf9 (Ras-associated domain family member 9), Tbx1 (T-box transcription factor 1) and interleukin (IL)-7. Characterisation of these genes may help to uncover unique functions of LECs as opposed to LSECs.

      Lymph and lymphatic drainage

      Hepatic lymph (lymphatic fluid) consists of sinusoidal plasma filtered into the space of Disse,
      • Poonkhum R.
      • Pisetpaisan K.
      • Wang B.J.
      • Anupunpisit V.
      • Ohtani Y.
      • Ohtani O.
      Origins and pathways of fluid entering sublobular lymphatic vessels in cat livers.
      an interstitial space between LSECs and hepatocytes, through fenestrae of LSECs. Although detailed hepatic lymph contents have not yet been specified, these contents potentially include cellular byproducts discharged from hepatic cells in the space of Disse (Fig. 1). Lymph is then thought to flow into lymphatic vessels located in 3 regions: portal, hepatic venous and sub-capsular areas.
      • Comparini L.
      Lymph vessels of the liver in man. Microscopic morphology and histotopography.
      Among them, lymphatic vessels in the portal tract are considered to be the primary site of hepatic lymph drainage, covering around 80% of the lymph produced by the liver.
      • Ritchie H.D.
      • Grindlay J.H.
      • Bollman J.L.
      Flow of lymph from the canine liver.
      Because the hepatic sinusoids are highly permeable and oncotic pressure along the sinusoids is negligible, fluid in the hepatic sinusoids can flow through the channels traversing the limiting plate to the interstitial space of the portal tract, following hydrostatic pressure gradients.
      Lymphatic capillaries in the portal tract coalesce into collecting lymphatic vessels surrounded by lymphatic muscle cells outside the liver.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      ,
      • Ohtani O.
      • Ohtani Y.
      Lymph circulation in the liver.
      ,
      • Barbier L.
      • Tay S.S.
      • McGuffog C.
      • Triccas J.A.
      • McCaughan G.W.
      • Bowen D.G.
      • et al.
      Two lymph nodes draining the mouse liver are the preferential site of DC migration and T cell activation.
      Lymphatic muscle cells covering collecting lymphatic vessels help to pump lymphatic fluid into regional lymph nodes known as hepatic or hilar lymph nodes located in the hepatic hilum. From these lymph nodes, lymphatic fluid flows to celiac lymph nodes through collecting lymphatic vessels and then drains to the cisterna chyli located at the lower end of the thoracic duct. Lymphatic capillaries running along the hepatic vein merge into 5-6 large lymphatic vessels, which traverse – along with the inferior vena cava – toward posterior mediastinal lymph nodes through the diaphragm. Lymphatic vessels along the hepatic capsule drain lymphatic fluid underneath the capsule of the convex surface of the liver to regional lymph nodes such as diaphragmatic lymph nodes in the thoracic region and then to mediastinal lymph nodes, similar to those along the hepatic vein.
      Hepatic lymphatic vessels are involved in fluid homeostasis, the immune system and lipid metabolism as they transport various immune cells, antigens and lipids to lymph nodes.
      While studies have suggested specific draining lymph nodes associated with lymphatic vessels in respective areas as mentioned above, the route of lymphatic drainage may be more complex and further analysis may be needed. Lymphatic vessels along the portal tract and the hepatic vein are called “the deep lymphatic system”, while those along the hepatic capsule are called “the superficial lymphatic system”.
      • Ohtani O.
      • Ohtani Y.
      Lymph circulation in the liver.
      A recent study
      • Frenkel N.C.
      • Poghosyan S.
      • Verheem A.
      • Padera T.P.
      • Rinkes I.
      • Kranenburg O.
      • et al.
      Liver lymphatic drainage patterns follow segmental anatomy in a murine model.
      that examined drainage patterns of the deep lymphatic system in the mouse liver by lymphangiography showed that hepatic lymphatic fluid was preferentially drained into regional hilar lymph nodes when it came from the right or left lobe. However, hepatic lymphatic fluid from the median lobe was mainly drained into mediastinal lymph nodes rather than hilar lymph nodes. These observations may suggest that the hepatic lymphatic drainage system is organized in a lobe-specific manner in mice.

      Lymphangiogenesis

      Pro- and anti-lymphangiogenic factors in the liver

      In adults, lymphatic vessels generally remain quiescent in normal conditions, with lymphangiogenesis occurring in pathological conditions, e.g. during tissue repair, inflammation and tumour development. Many cytokines and growth factors have been reported to promote or inhibit lymphangiogenesis in other organs. Among them, those cytokines and factors detected in the liver in physiological and pathophysiological conditions (thus not necessarily studied for lymphangiogenesis) are listed in Table S2 as potential pro-and anti-lymphangiogenic factors in the liver. Cellular sources of these factors have not been fully identified in the liver.

      VEGF-C/D and VEGFR3 signalling

      Signals mediated by members of the VEGF and VEGFR families are known to play central roles in angiogenesis and lymphangiogenesis (Fig. 3).
      • Leung D.W.
      • Cachianes G.
      • Kuang W.J.
      • Goeddel D.V.
      • Ferrara N.
      Vascular endothelial growth factor is a secreted angiogenic mitogen.
      • Alitalo K.
      • Carmeliet P.
      Molecular mechanisms of lymphangiogenesis in health and disease.
      • Ferrara N.
      Vascular endothelial growth factor: basic science and clinical progress.
      VEGF-A binds to VEGFR1/Flt1 and VEGFR2/KDR
      • de Vries C.
      • Escobedo J.A.
      • Ueno H.
      • Houck K.
      • Ferrara N.
      • Williams L.T.
      The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.
      ,
      • Terman B.I.
      • Dougher-Vermazen M.
      • Carrion M.E.
      • Dimitrov D.
      • Armellino D.C.
      • Gospodarowicz D.
      • et al.
      Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.
      and mediates angiogenesis, while VEGF-B and placental growth factor bind only to VEGFR1/Flt1.
      • Alitalo K.
      • Carmeliet P.
      Molecular mechanisms of lymphangiogenesis in health and disease.
      ,
      • Olofsson B.
      • Korpelainen E.
      • Pepper M.S.
      • Mandriota S.J.
      • Aase K.
      • Kumar V.
      • et al.
      Vascular endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates plasminogen activator activity in endothelial cells.
      VEGF-C and D bind strongly to VEGFR3/Flt4, leading to lymphangiogenesis, while they also bind very weakly to VEGFR2/KDR.
      • Alitalo K.
      • Carmeliet P.
      Molecular mechanisms of lymphangiogenesis in health and disease.
      ,
      • Achen M.G.
      • Jeltsch M.
      • Kukk E.
      • Makinen T.
      • Vitali A.
      • Wilks A.F.
      • et al.
      Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4).
      VEGF-C and D are initially synthesised as precursor forms that subsequently undergo proteolytic cleavage, removing their C- and N-terminal propeptides for induction of lymphangiogenesis.
      • Alitalo K.
      • Carmeliet P.
      Molecular mechanisms of lymphangiogenesis in health and disease.
      Currently, 5 different proteases are known to cleave VEGF-C, including plasmin, ADAMTS3 (A distintegrin and metalloprotease with thrombospondin motifs-3), prostate-specific antigen, cathepsin D and thrombin. All these proteases except for ADAMTS3 can also activate VEGF-D. The usual VEGF-C cleaving enzyme is ADAMTS3, which requires binding to its cofactor, CCBE1 (collagen and calcium binding EGF domains 1), for successful pro-VEGF-C activation.
      • Bui H.M.
      • Enis D.
      • Robciuc M.R.
      • Nurmi H.J.
      • Cohen J.
      • Chen M.
      • et al.
      Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD.
      ,
      • Le Guen L.
      • Karpanen T.
      • Schulte D.
      • Harris N.C.
      • Koltowska K.
      • Roukens G.
      • et al.
      Ccbe1 regulates Vegfc-mediated induction of Vegfr3 signaling during embryonic lymphangiogenesis.
      The inability of ADAMTS3-CCBE1 to activate VEGF-D
      • Bui H.M.
      • Enis D.
      • Robciuc M.R.
      • Nurmi H.J.
      • Cohen J.
      • Chen M.
      • et al.
      Proteolytic activation defines distinct lymphangiogenic mechanisms for VEGFC and VEGFD.
      may suggest that biological events induced by VEGF-C and D are tissue or context dependent.
      Figure thumbnail gr3
      Fig. 3VEGFs and VEGFRs in angiogenesis and lymphangiogenesis.
      VEGF is a potent mediator of both angiogenesis and lymphangiogenesis. All members of the VEGF family induce cellular responses by binding to specific VEGF receptors with tyrosine kinases, leading them to dimerise and activate through phosphorylation. VEGF-C and D bind strongly to VEGFR3/Flt4 and induce lymphangiogenesis, while they also bind very weakly to VEGFR2/KDR. VEGF-A binds to VEGFR1/Flt1 and VEGFR2/KDR and mediates angiogenesis. VEGF-B and PIGF bind only to VEGFR1/Flt1. Thick and thin arrows indicate strong and weak binding, respectively. VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

      Lymphatics and the immune system

      LECs express various chemokines and recruit immune cells. The most-studied chemokine in this regard is C–C motif chemokine ligand 21 (CCL21), a lymphoid homing chemokine.
      • Lo J.C.
      • Chin R.K.
      • Lee Y.
      • Kang H.S.
      • Wang Y.
      • Weinstock J.V.
      • et al.
      Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues.
      CCL21 guides dendritic cells (DCs), which express its receptor C–C chemokine receptor type 7 (CCR7), and other CCR7-expressing immune cells, such as T-cell subsets (naïve, memory and regulatory T cells) and neutrophils, to lymph nodes via lymphatic vessels.
      • Unsoeld H.
      • Mueller K.
      • Schleicher U.
      • Bogdan C.
      • Zwirner J.
      • Voehringer D.
      • et al.
      Abrogation of CCL21 chemokine function by transgenic over-expression impairs T cell immunity to local infections.
      • Beauvillain C.
      • Cunin P.
      • Doni A.
      • Scotet M.
      • Jaillon S.
      • Loiry M.L.
      • et al.
      CCR7 is involved in the migration of neutrophils to lymph nodes.
      • Saeki H.
      • Moore A.M.
      • Brown M.J.
      • Hwang S.T.
      Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.
      LECs also express intracellular adhesion molecule 1
      • Podgrabinska S.
      • Kamalu O.
      • Mayer L.
      • Shimaoka M.
      • Snoeck H.
      • Randolph G.J.
      • et al.
      Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac-1/ICAM-1-dependent mechanism.
      and C-X3-C motif chemokine ligand 1 to guide DCs to lymphatic vessels in inflamed skin.
      • Johnson L.A.
      • Jackson D.G.
      The chemokine CX3CL1 promotes trafficking of dendritic cells through inflamed lymphatics.
      Further, LEC-derived sphingosine-1-phosphate (S1P) has been identified as a critical lipid mediator that interacts with the S1P receptor 1 (S1P1) on T cells to promote their egression from lymph nodes
      • Schwab S.R.
      • Cyster J.G.
      Finding a way out: lymphocyte egress from lymphoid organs.
      and the spleen.
      • Lucaciu A.
      • Kuhn H.
      • Trautmann S.
      • Ferreiros N.
      • Steinmetz H.
      • Pfeilschifter J.
      • et al.
      A sphingosine 1-phosphate gradient is linked to the cerebral recruitment of T helper and regulatory T helper cells during acute ischemic stroke.
      Besides T-cell migration, LECs also regulate T-cell function. Human and murine LECs express major histocompatibility complex class I and class II molecules and may directly induce T-cell tolerance, which prevents self-activation of T cells in response to innocuous proteins and self-antigens under normal conditions.
      • Rouhani S.J.
      • Eccles J.D.
      • Riccardi P.
      • Peske J.D.
      • Tewalt E.F.
      • Cohen J.N.
      • et al.
      Roles of lymphatic endothelial cells expressing peripheral tissue antigens in CD4 T-cell tolerance induction.
      • Vokali E.
      • Yu S.S.
      • Lund A.W.
      • Duraes F.V.
      • Hirosue S.
      • Raghavan V.R.
      • Nembrini C.
      • Thomas S.N.
      • et al.
      VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics.
      • Malhotra D.
      • Fletcher A.L.
      • Astarita J.
      • Lukacs-Kornek V.
      • Tayalia P.
      • Gonzalez S.F.
      • et al.
      Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.
      Further, multiple peripheral tissue antigens expressed in LECs are known to play an important role in inducing T-cell tolerance and mediating deletion of self-reactive CD8+ T cells. LECs also secrete immunoregulatory factors, including transforming growth factor-β, IDO (indoleamine-2,3-dioxygenase) and nitric oxide, to suppress T-cell activation.
      • Malhotra D.
      • Fletcher A.L.
      • Astarita J.
      • Lukacs-Kornek V.
      • Tayalia P.
      • Gonzalez S.F.
      • et al.
      Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.
      ,
      • Lukacs-Kornek V.
      • Malhotra D.
      • Fletcher A.L.
      • Acton S.E.
      • Elpek K.G.
      • Tayalia P.
      • et al.
      Regulated release of nitric oxide by nonhematopoietic stroma controls expansion of the activated T cell pool in lymph nodes.
      While lymphatic vessel numbers are known to increase (via lymphangiogenesis) in many liver diseases, the mechanism of lymphangiogenesis in the liver remains poorly understood.
      These observations of LEC’s immunomodulatory function in other organs could be applicable to hepatic LECs and help to uncover the role of lymphatic vessels in liver functions. In chronic liver diseases, lymphatics play a role in immune cell trafficking. An increase in the number of CCL21-expressing LECs has been reported in the livers of patients with non-alcoholic steatohepatitis (NASH).
      • Tamburini B.A.J.
      • Finlon J.M.
      • Gillen A.E.
      • Kriss M.S.
      • Riemondy K.A.
      • Fu R.
      • et al.
      Chronic liver disease in humans causes expansion and differentiation of liver lymphatic endothelial cells.
      Although the relationship between increased levels of CCL21 and hepatic lymphatics has not been investigated, other studies also reported an increase in hepatic CCL21 expression in primary biliary cholangitis (PBC), primary sclerosing cholangitis and alcohol-associated liver disease.
      • Grant A.J.
      • Goddard S.
      • Ahmed-Choudhury J.
      • Reynolds G.
      • Jackson D.G.
      • Briskin M.
      • et al.
      Hepatic expression of secondary lymphoid chemokine (CCL21) promotes the development of portal-associated lymphoid tissue in chronic inflammatory liver disease.
      ,
      • Borchers A.T.
      • Shimoda S.
      • Bowlus C.
      • Keen C.L.
      • Gershwin M.E.
      Lymphocyte recruitment and homing to the liver in primary biliary cirrhosis and primary sclerosing cholangitis.
      Similarly, S1P signalling has been shown to be a key player in metabolic diseases and various liver pathologies including NAFLD, NASH and liver fibrosis.
      • Kleuser B.
      Divergent role of sphingosine 1-phosphate in liver health and disease.
      The underlying mechanisms behind S1P’s role in the regulation of hepatic glucose and lipid metabolism in hepatocytes and in the activation of hepatic stellate cells has been explored.
      • Kleuser B.
      Divergent role of sphingosine 1-phosphate in liver health and disease.
      ,
      • Frej C.
      • Linder A.
      • Happonen K.E.
      • Taylor F.B.
      • Lupu F.
      • Dahlbäck B.
      Sphingosine 1-phosphate and its carrier apolipoprotein M in human sepsis and in Escherichia coli sepsis in baboons.
      However, it will be interesting to examine the pathological significance of S1P’s effect on hepatic lymphatics.

      Lymphatics in liver diseases

      Hepatic lymphangiogenesis

      Lymphangiogenesis with increased and enlarged lymphatic vessels was reported in rat livers with carbon tetrachloride (CCl4)-induced fibrosis/cirrhosis,
      • Vollmar B.
      • Wolf B.
      • Siegmund S.
      • Katsen A.D.
      • Menger M.D.
      Lymph vessel expansion and function in the development of hepatic fibrosis and cirrhosis.
      as well as in patients with chronic viral hepatitis/cirrhosis.
      • Yamauchi Y.
      • Michitaka K.
      • Onji M.
      Morphometric analysis of lymphatic and blood vessels in human chronic viral liver diseases.
      In 2 rat models of portal hypertension (portacaval shunt and partial portal vein ligation), upregulation of VEGFR3 expression was observed. This observation leads us to speculate that lymphangiogenesis is probably occurring in these models of portal hypertension as well.
      • Guérin F.
      • Wagner M.
      • Liné A.
      • Zappa M.
      • Fasseu M.
      • Paradis V.
      • et al.
      Hepatic proliferation and angiogenesis markers are increased after portal deprivation in rats: a study of molecular, histological and radiological changes.
      In addition, liver specimens from patients with early stage PBC showed an increase in the number and the luminal area of lymphatic vessels, indicating that lymphangiogenesis occurred even in the early stages of PBC.
      • Yamauchi Y.
      • Ikeda R.
      • Michitaka K.
      • Hiasa Y.
      • Horiike N.
      • Onji M.
      Morphometric analysis of lymphatic vessels in primary biliary cirrhosis.
      Further, microarray analysis revealed a 4-fold increase in VEGF-D expression in endothelial cells isolated from cirrhotic rat livers (CCl4 model) compared with control rat livers,
      • Tugues S.
      • Morales-Ruiz M.
      • Fernandez-Varo G.
      • Ros J.
      • Arteta D.
      • Munoz-Luque J.
      • et al.
      Microarray analysis of endothelial differentially expressed genes in liver of cirrhotic rats.
      implicating VEGF-D in the hepatic lymphangiogenesis observed in these cirrhotic rat livers.

      Hepatic lymph production in cirrhosis with portal hypertension

      Resistance to sinusoidal blood flow increases in cirrhotic livers because of the architectural deformations that result from massive fibrosis. Consequently, hydrostatic pressure in the sinusoids is elevated, and plasma components filtrated through the sinusoids (which form lymph) increase.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      ,
      • Chung C.
      • Iwakiri Y.
      The lymphatic vascular system in liver diseases: its role in ascites formation.
      This mechanism is supported by observations in which lowering portal venous pressure by portocaval shunt surgery decreased the lymph flow rate in dogs
      • Orloff M.J.
      • Goodhead B.
      • Windsor C.W.
      • Musicant M.E.
      • Annetts D.L.
      Effect of portacaval shunts on lymph flow in the thoracic duct. Experiments with normal dogs and dogs with cirrhosis and ascites.
      and patients
      • Dumont A.E.
      • Mulholland J.H.
      Flow rate and composition of thoracic-duct lymph in patients with cirrhosis.
      ,
      • Witte M.H.
      • Dumont A.E.
      • Cole W.R.
      • Witte C.L.
      • Kintner K.
      Lymph circulation in hepatic cirrhosis: effect of portacaval shunt.
      with cirrhosis. Transjugular intrahepatic portosystemic shunts were also shown to have the same effect on thoracic duct lymph flow.
      • Vignaux O.
      • Gouya H.
      • Dousset B.
      • Mazuir E.
      • Buffet C.
      • Calmus Y.
      • et al.
      Refractory chylothorax in hepatic cirrhosis: successful treatment by transjugular intrahepatic portosystemic shunt.
      In cirrhosis, hepatic lymph production significantly increases in both humans
      • Dumont A.E.
      • Mulholland J.H.
      Flow rate and composition of thoracic-duct lymph in patients with cirrhosis.
      and rats.
      • Barrowman J.A.
      • Granger D.N.
      Effects of experimental cirrhosis on splanchnic microvascular fluid and solute exchange in the rat.
      ,
      • Nix J.T.
      • Flock E.V.
      • Bollman J.L.
      Influence of cirrhosis on proteins of cisternal lymph.
      It was reported that patients with cirrhosis had 3–6-fold higher lymph flow rates in the thoracic duct (whose diameter was increased by 2–4-fold) than patients without cirrhosis.
      • Dumont A.E.
      • Mulholland J.H.
      Flow rate and composition of thoracic-duct lymph in patients with cirrhosis.
      In this study, the thoracic duct was cannulated to measure the flow rate and compositions of lymph. One study reported that hepatic lymph flow (measured by cannulating the hepatic lymph trunk) was nearly 30-fold higher in cirrhotic rats with portal hypertension compared to control rats,
      • Barrowman J.A.
      • Granger D.N.
      Effects of experimental cirrhosis on splanchnic microvascular fluid and solute exchange in the rat.
      while another study estimated the increase to be nearer 6-fold using a similar method of measurement.
      • Nix J.T.
      • Flock E.V.
      • Bollman J.L.
      Influence of cirrhosis on proteins of cisternal lymph.
      This discrepancy in the magnitude of flow could be due to the timing of measurements or the severity of cirrhosis. Increased collateral lymph flow in the mediastinum and oesophagus results in bloody lymph in the thoracic duct due to communication between lymphatic vessels and veins.
      • Dumont A.E.
      • Witte C.L.
      • Witte M.H.
      • Cole W.R.
      Origin of red blood cells in thoracic duct lymph in hepatic cirrhosis.

      Ascites formation

      Ascites formation in association with cirrhosis is one of the most recognised clinical manifestations of disorders of the lymphatic system.
      • Chung C.
      • Iwakiri Y.
      The lymphatic vascular system in liver diseases: its role in ascites formation.
      Currently, the most accepted theory of ascites formation is the “forward theory” (Fig. 4).
      • De Franchis R.
      • Salerno F.
      Pathogenesis of ascites and predictors of resistance to therapy.
      • Arroyo V.
      • Colmenero J.
      Ascites and hepatorenal syndrome in cirrhosis: pathophysiological basis of therapy and current management.
      • Gordon F.D.
      Ascites.
      According to this theory, splanchnic arterial vasodilation caused by portal hypertension results in underfilling of the splanchnic arterial circulation or hypovolemia. In moderate stages, hypovolemia is compensated by renal retention of sodium and water.
      • Schrier R.W.
      • Arroyo V.
      • Bernardi M.
      • Epstein M.
      • Henriksen J.H.
      • Rodes J.
      Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis.
      However, severe portal hypertension and splanchnic arterial vasodilation make sodium and water retention persistent and lead to leaking fluid into the peritoneal cavity. In cirrhosis, decreased oncotic pressure caused by hypoalbuminemia also promotes fluid leakage into the peritoneal cavity.
      • Mankin H.
      • Lowell A.
      Osmotic factors influencing the formation of ascites in patients with cirrhosis of the liver.
      As described above, increased intrahepatic vascular resistance due to cirrhosis results in elevated hydrostatic pressure and the consequent increase in hepatic lymph production as well.
      VEGF-C and D bind to VEGFR3/Flt4, leading to lymphangiogenesis, while VEGF-A binds to VEGFR1/Flt1 and VEGFR2/KDR, mediating angiogenesis.
      Figure thumbnail gr4
      Fig. 4Mechanisms of ascites formation in cirrhosis with portal hypertension.
      Elevated hydrostatic pressure in the sinusoids due to cirrhosis causes an increased production of lymph. It is thought that ascitic fluid starts to accumulate when capsular or superficial lymphatics of the liver rupture and hepatic lymph with a high protein concentration leaks into the peritoneal cavity. This lymph leakage from the surface of the liver is known as the so-called ‘weeping liver’. When the total fluid flux into the peritoneal cavity exceeds the lymph-draining capacity of the peritoneum, ascites forms. According to the “forward theory”, portal hypertension causes excessive arterial vasodilation in the splanchnic arterial circulation, leading to the underfilling of the arterial circulation or hypovolemia. Splanchnic arterial vasodilation makes sodium and water retention persistent and leads to leaking fluid into the peritoneal cavity and accumulation of ascites.
      Levitt et al. looked into the local mechanism of ascites formation in the peritoneal cavity and developed a quantitative model.
      • Levitt D.G.
      • Levitt M.D.
      Quantitative modeling of the physiology of ascites in portal hypertension.
      Equations they formulated described the fluid flux based on hydrostatic pressure and colloid osmotic pressure, and were validated by actual parameters obtained from animal experiments and clinical data. This model demonstrates that an elevation of portal pressure itself does not cause appreciable ascites. Ascitic fluid starts to accumulate when capsular or superficial lymphatics of the liver rupture and hepatic lymph with a high protein concentration leaks into the peritoneal cavity. This ascitic fluid has high colloidal pressure and causes transport of water from the mesentery to the peritoneal cavity, which results in further increases of ascitic fluid. The lymph leakage from the surface of the liver has been observed directly by laparoscopy and is known as the so-called ‘weeping liver’
      • Witte C.L.
      • Witte M.H.
      • Dumont A.E.
      • Frist J.
      • Cole W.R.
      Lymph protein in hepatic cirrhosis and experimental hepatic and portal venous hypertension.
      ,
      • Dumont A.E.
      • Mulholland J.H.
      Alterations in thoracic duct lymph flow in hepatic cirrhosis: significance in portal hypertension.
      ; no studies have yet quantified this lymph leakage. It is thought that ascites forms when the total fluid flux into the peritoneal cavity exceeds the lymph draining capacity of the peritoneum, which is approximately 50 ml/hour at most.
      • Leak L.V.
      • Rahil K.
      Permeability of the diaphragmatic mesothelium: the ultrastructural basis for "stomata".
      • Henriksen J.H.
      • Lassen N.A.
      • Parving H.H.
      • Winkler K.
      Filtration as the main transport mechanism of protein exchange between plasma and the peritoneal cavity in hepatic cirrhosis.
      • Henriksen J.H.
      • Siemssen O.
      • Krintel J.J.
      • Malchow-Møller A.
      • Bendtsen F.
      • Ring-Larsen H.
      Dynamics of albumin in plasma and ascitic fluid in patients with cirrhosis.
      • Sanyal A.J.
      • Bosch J.
      • Blei A.
      • Arroyo V.
      Portal hypertension and its complications.

      Malignant tumours

      Lymphatic vessels play a pivotal role in the pathogenesis of malignant tumours as a pathway through which tumour cells spread.
      • Stacker S.A.
      • Williams S.P.
      • Karnezis T.
      • Shayan R.
      • Fox S.B.
      • Achen M.G.
      Lymphangiogenesis and lymphatic vessel remodelling in cancer.
      ,
      • Oliver G.
      • Kipnis J.
      • Randolph G.J.
      • Harvey N.L.
      The lymphatic vasculature in the 21(st) century: novel functional roles in homeostasis and disease.
      The incidence of lymph node metastasis differs among malignant tumours. In liver cancer, lymph node metastasis was observed in 5.1% of hepatocellular carcinoma (HCC) cases and 45.1% of intrahepatic cholangiocarcinoma (ICC) cases.
      • Sun H.C.
      • Zhuang P.Y.
      • Qin L.X.
      • Ye Q.H.
      • Wang L.
      • Ren N.
      • et al.
      Incidence and prognostic values of lymph node metastasis in operable hepatocellular carcinoma and evaluation of routine complete lymphadenectomy.
      The prognosis of tumour-bearing patients with lymph node metastasis is worse than the cases without lymph node metastasis. In ICC, the lymphatic vessel density of surgically resected ICCs increased as the extent of malignancy worsened,
      • Aishima S.
      • Nishihara Y.
      • Iguchi T.
      • Taguchi K.
      • Taketomi A.
      • Maehara Y.
      • et al.
      Lymphatic spread is related to VEGF-C expression and D2-40-positive myofibroblasts in intrahepatic cholangiocarcinoma.
      and lymphatic vessel density was correlated with higher incidences of lymphatic metastasis.
      • Thelen A.
      • Scholz A.
      • Weichert W.
      • Wiedenmann B.
      • Neuhaus P.
      • Gessner R.
      • et al.
      Tumor-associated angiogenesis and lymphangiogenesis correlate with progression of intrahepatic cholangiocarcinoma.
      As mentioned, CCL21 in LECs recruits CCR7-expressing DCs toward lymphatic vessels and facilitates their egression from the liver.
      • Förster R.
      • Davalos-Misslitz A.C.
      • Rot A.
      CCR7 and its ligands: balancing immunity and tolerance.
      However, CCR7 is also expressed by a variety of malignant tumours, and the CCL21–CCR7 axis is considered a causal factor for lymph node metastasis.
      • Günther K.
      • Leier J.
      • Henning G.
      • Dimmler A.
      • Weissbach R.
      • Hohenberger W.
      • et al.
      Prediction of lymph node metastasis in colorectal carcinoma by expressionof chemokine receptor CCR7.
      • Cabioglu N.
      • Yazici M.S.
      • Arun B.
      • Broglio K.R.
      • Hortobagyi G.N.
      • Price J.E.
      • et al.
      CCR7 and CXCR4 as novel biomarkers predicting axillary lymph node metastasis in T1 breast cancer.
      • Mashino K.
      • Sadanaga N.
      • Yamaguchi H.
      • Tanaka F.
      • Ohta M.
      • Shibuta K.
      • et al.
      Expression of chemokine receptor CCR7 is associated with lymph node metastasis of gastric carcinoma.
      • Irino T.
      • Takeuchi H.
      • Matsuda S.
      • Saikawa Y.
      • Kawakubo H.
      • Wada N.
      • et al.
      CC-Chemokine receptor CCR7: a key molecule for lymph node metastasis in esophageal squamous cell carcinoma.
      • Zhang S.
      • Wang H.
      • Xu Z.
      • Bai Y.
      • Xu L.
      Lymphatic metastasis of NSCLC involves chemotaxis effects of lymphatic endothelial cells through the CCR7-CCL21 Axis modulated by TNF-α.
      • Pang M.F.
      • Georgoudaki A.M.
      • Lambut L.
      • Johansson J.
      • Tabor V.
      • Hagikura K.
      • et al.
      TGF-β1-induced EMT promotes targeted migration of breast cancer cells through the lymphatic system by the activation of CCR7/CCL21-mediated chemotaxis.
      A positive correlation between expression levels of CCR7 and lymph node metastasis was reported in patients with HCC.
      • Schimanski C.C.
      • Bahre R.
      • Gockel I.
      • Junginger T.
      • Simiantonaki N.
      • Biesterfeld S.
      • et al.
      Chemokine receptor CCR7 enhances intrahepatic and lymphatic dissemination of human hepatocellular cancer.
      In addition, many malignant tumours are known to secrete lymphangiogenic factors, such as VEGF-C and VEGF-D, and promote lymphangiogenesis in adjacent tissues, which helps tumour cells to metastasise to lymph nodes.
      • Das S.
      • Skobe M.
      Lymphatic vessel activation in cancer.
      Many studies have demonstrated that tumour-associated macrophages play a vital role in lymphangiogenesis in malignant tumours by secreting VEGF-C and VEGF-D.
      • Schoppmann S.F.
      • Birner P.
      • Stockl J.
      • Kalt R.
      • Ullrich R.
      • Caucig C.
      • et al.
      Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis.
      • Skobe M.
      • Hamberg L.M.
      • Hawighorst T.
      • Schirner M.
      • Wolf G.L.
      • Alitalo K.
      • et al.
      Concurrent induction of lymphangiogenesis, angiogenesis, and macrophage recruitment by vascular endothelial growth factor-C in melanoma.
      • Iwata C.
      • Kano M.R.
      • Komuro A.
      • Oka M.
      • Kiyono K.
      • Johansson E.
      • et al.
      Inhibition of cyclooxygenase-2 suppresses lymph node metastasis via reduction of lymphangiogenesis.
      • Schoppmann S.F.
      • Fenzl A.
      • Nagy K.
      • Unger S.
      • Bayer G.
      • Geleff S.
      • et al.
      VEGF-C expressing tumor-associated macrophages in lymph node positive breast cancer: impact on lymphangiogenesis and survival.
      In ICC, VEGF-C expression was associated with a higher rate of lymph node metastasis and worse prognosis.
      • Aishima S.
      • Nishihara Y.
      • Iguchi T.
      • Taguchi K.
      • Taketomi A.
      • Maehara Y.
      • et al.
      Lymphatic spread is related to VEGF-C expression and D2-40-positive myofibroblasts in intrahepatic cholangiocarcinoma.
      ,
      • Park B.K.
      • Paik Y.H.
      • Park J.Y.
      • Park K.H.
      • Bang S.
      • Park S.W.
      • et al.
      The clinicopathologic significance of the expression of vascular endothelial growth factor-C in intrahepatic cholangiocarcinoma.
      In HCC, VEGF-C expression was shown to correlate positively with the size of tumours and the number of sites of extrahepatic metastasis and negatively with disease-free survival time.
      • Yamaguchi R.
      • Yano H.
      • Nakashima O.
      • Akiba J.
      • Nishida N.
      • Kurogi M.
      • et al.
      Expression of vascular endothelial growth factor-C in human hepatocellular carcinoma.
      Plasma VEGF-C levels in patients undergoing liver transplantation for HCC were negatively associated with both their disease-free survival rates and overall survival rates.
      • Duda D.G.
      • Dima S.O.
      • Cucu D.
      • Sorop A.
      • Klein S.
      • Ancukiewicz M.
      • et al.
      Potential circulating biomarkers of recurrence after hepatic resection or liver transplantation in hepatocellular carcinoma patients.
      A recent study demonstrated that ICC-derived platelet-derived growth factor D (PDGF-D) recruited and activated cancer-associated fibroblasts (CAFs) in stromal components adjacent to ICC. Activated CAFs then secreted VEGF-A and VEGF-C, inducing lymphangiogenesis and thereby promoting lymph node metastasis.
      • Cadamuro M.
      • Brivio S.
      • Mertens J.
      • Vismara M.
      • Moncsek A.
      • Milani C.
      • et al.
      Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.
      The study also showed that a VEGFR3 antagonist reduced tumour-associated lymphangiogenesis in a xenograft model using SCID (severe combined immunodeficiency) mice transplanted with PDGF-D-secreting human ICC cell lines. No selective agents that specifically suppress lymphangiogenesis have been approved for clinical use.
      • Yamakawa M.
      • Doh S.J.
      • Santosa S.M.
      • Montana M.
      • Qin E.C.
      • Kong H.
      • et al.
      Potential lymphangiogenesis therapies: learning from current antiangiogenesis therapies-A review.
      A phase I clinical trial of a VEGF-C neutralising antibody (VGX-100) for adult patients with advanced or metastatic solid tumours (NCT01514123) was completed in 2014, but a phase II trial has not been opened yet.
      • Tampellini M.
      • Sonetto C.
      • Scagliotti G.V.
      Novel anti-angiogenic therapeutic strategies in colorectal cancer.
      ,
      • Falchook G.S.
      • Goldman J.W.
      • Desai J.
      • Leitch I.
      • Hong D.S.
      • Subbiah V.
      • et al.
      A first-in-human phase I study of VGX-100, a selective anti-VEGF-C antibody, alone and in combination with bevacizumab in patients with advanced solid tumors.
      A phase I clinical trial of an anti-VEGFR3 monoclonal antibody (LY3022856/IMC-3C5) for patients with advanced solid tumours demonstrated minimal antitumour effect.
      • Saif M.W.
      • Knost J.A.
      • Chiorean E.G.
      • Kambhampati S.R.
      • Yu D.
      • Pytowski B.
      • et al.
      Phase 1 study of the anti-vascular endothelial growth factor receptor 3 monoclonal antibody LY3022856/IMC-3C5 in patients with advanced and refractory solid tumors and advanced colorectal cancer.
      Ascites formation in association with cirrhosis is one of the most recognised clinical manifestations of disorders of the lymphatic system.
      Conversely, interesting findings that VEGF-C/VEGFR3-driven lymphangiogenesis enhances the antitumour effect of immunotherapy have recently been reported in some tumours. In one study, VEGF-C-overexpressing mice were inoculated intradermally with a melanoma cell line to evaluate the potential synergism of a VEGFR3 blocking antibody alongside adaptive immunotherapy.
      • Fankhauser M.
      • Broggi M.A.S.
      • Potin L.
      • Bordry N.
      • Jeanbart L.
      • Lund A.W.
      • et al.
      Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma.
      Contrary to expectations, blocking VEGFR3 suppressed the effect of immunotherapy and worsened the survival rate. It was demonstrated that VEGF-C overexpression facilitated intratumoral lymphangiogenesis, upregulated intratumoral CCL21 expression and promoted infiltration of CCR7-expressing naïve T cells into tumours. Conversely, VEGFR3 blocking inhibited intratumoral lymphangiogenesis, decreased intratumoral CCL21 expression and suppressed the number of intratumoral naïve T cells, which may have contributed to decreased sensitivity to immunotherapy. It was also shown that serum VEGF-C concentrations positively correlated with both T-cell activation after peptide vaccination and overall survival rates after checkpoint blockade in patients with metastatic melanoma.
      • Fankhauser M.
      • Broggi M.A.S.
      • Potin L.
      • Bordry N.
      • Jeanbart L.
      • Lund A.W.
      • et al.
      Tumor lymphangiogenesis promotes T cell infiltration and potentiates immunotherapy in melanoma.
      Another study using a mouse model of glioblastoma demonstrated that adeno-associated virus-mediated VEGF-C gene transfer promoted meningeal lymphatic drainage and improved survival rates in a mouse model of glioblastoma.
      • Song E.
      • Mao T.
      • Dong H.
      • Boisserand L.S.B.
      • Antila S.
      • Bosenberg M.
      • et al.
      VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.
      Ligation of deep cervical lymph nodes, to which cerebrospinal fluid is drained through meningeal lymphatic vessels, as well as depletion of CD4-or CD8-positive T cells negated the beneficial effect of VEGF-C, indicating that the antitumour effect of VEGF-C was mediated by T cells that migrated to lymph nodes. It was also shown that VEGF-C gene transfer enhanced the effect of anti-PD-L1 antibody treatment. These findings indicate that tumour-associated lymphatic vessels not only act as a pathway for tumour dissemination, but also as an integral part of T cell-mediated antitumour immunity.

      Liver transplantation

      Graft rejection is one of the most serious concerns in solid organ transplantation. Alloimmunity (responses to non-self-antigens from the same species) is established once alloantigens of the graft are drained into secondary lymphoid organs, through lymphatic vessels, and encounter T lymphocytes.
      • Liao S.
      • von der Weid P.Y.
      Lymphatic system: an active pathway for immune protection.
      Therefore, the potential role of lymphangiogenesis in graft rejection has received considerable attention.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      Post-transplant lymphangiogenesis in grafts was associated with acute cellular graft rejection in various organs (kidney,
      • Kerjaschki D.
      • Regele H.M.
      • Moosberger I.
      • Nagy-Bojarski K.
      • Watschinger B.
      • Soleiman A.
      • et al.
      Lymphatic neoangiogenesis in human kidney transplants is associated with immunologically active lymphocytic infiltrates.
      heart
      • Geissler H.J.
      • Dashkevich A.
      • Fischer U.M.
      • Fries J.W.
      • Kuhn-Regnier F.
      • Addicks K.
      • et al.
      First year changes of myocardial lymphatic endothelial markers in heart transplant recipients.
      and lung
      • Dashkevich A.
      • Heilmann C.
      • Kayser G.
      • Germann M.
      • Beyersdorf F.
      • Passlick B.
      • et al.
      Lymph angiogenesis after lung transplantation and relation to acute organ rejection in humans.
      ) in humans. However, in a rat model of liver transplantation, increased post-transplant lymphangiogenesis in grafts was associated with long-term survival (>90 days) of recipients. In addition, in rats that had failed to graft by 11 days, lymphatic vessels had disappeared from severely rejected areas. These observations suggest that lymphangiogenesis may play an important role in mitigating inflammation, at least in the early stage of liver transplantation.
      • Ishii E.
      • Shimizu A.
      • Kuwahara N.
      • Arai T.
      • Kataoka M.
      • Wakamatsu K.
      • et al.
      Lymphangiogenesis associated with acute cellular rejection in rat liver transplantation.
      This difference between the liver and other organs might be attributable in part to hepatic immune tolerance.
      • Dai H.
      • Zheng Y.
      • Thomson A.W.
      • Rogers N.M.
      Transplant tolerance induction: insights from the liver.
      Hepatic immune tolerance was first reported in 1969, when it was shown that liver allotransplantation did not lead to rejection of the second allograft transplanted from the same donor.
      • Calne R.Y.
      • Sells R.A.
      • Pena J.R.
      • Davis D.R.
      • Millard P.R.
      • Herbertson B.M.
      • et al.
      Induction of immunological tolerance by porcine liver allografts.
      In liver transplantation, human leukocyte antigen mismatch is not a problem,
      • Lan X.
      • Zhang M.M.
      • Pu C.L.
      • Guo C.B.
      • Kang Q.
      • Li Y.C.
      • et al.
      Impact of human leukocyte antigen mismatching on outcomes of liver transplantation: a meta-analysis.
      and immunosuppressant requirements are more mild compared with other organ transplantations.
      • Rodríguez-Perálvarez M.
      • Germani G.
      • Papastergiou V.
      • Tsochatzis E.
      • Thalassinos E.
      • Luong T.V.
      • et al.
      Early tacrolimus exposure after liver transplantation: relationship with moderate/severe acute rejection and long-term outcome.
      While the mechanism of hepatic immune tolerance is not fully understood, unique phenotypes of liver resident cells may play a role. For example, it was reported that Kupffer cells suppressed T-cell proliferation through secretion of nitric oxide in response to interferon-γ
      • Roland C.R.
      • Walp L.
      • Stack R.M.
      • Flye M.W.
      Outcome of Kupffer cell antigen presentation to a cloned murine Th1 lymphocyte depends on the inducibility of nitric oxide synthase by IFN-gamma.
      and that LSECs negatively regulated activated T cells via expression of LSEC-specific lectin that recognises these activated T cells.
      • Tang L.
      • Yang J.
      • Liu W.
      • Tang X.
      • Chen J.
      • Zhao D.
      • et al.
      Liver sinusoidal endothelial cell lectin, LSECtin, negatively regulates hepatic T-cell immune response.
      In addition, different responses of hepatic DCs compared to other DCs may contribute to immune tolerant environments in the liver. One study demonstrated that hepatic DCs secreted the anti-inflammatory cytokine IL10 upon Toll-like receptor 4 stimulation, while peripheral DCs secreted multiple proinflammatory cytokines.
      • Bamboat Z.M.
      • Stableford J.A.
      • Plitas G.
      • Burt B.M.
      • Nguyen H.M.
      • Welles A.P.
      • et al.
      Human liver dendritic cells promote T cell hyporesponsiveness.
      This study also showed that CD4-positive T cells initially stimulated by hepatic DCs were less responsive to restimulation than those T cells pre-stimulated by blood DCs. Another study demonstrated that hepatic DCs secreted less inflammatory cytokine IL12 than spleen DCs upon activation by Toll-like receptor 9 ligand.
      • Abe M.
      • Tokita D.
      • Raimondi G.
      • Thomson A.W.
      Endotoxin modulates the capacity of CpG-activated liver myeloid DC to direct Th1-type responses.
      This study also showed that T cells stimulated by hepatic DCs secreted less interferon-γ than those stimulated by spleen DCs. Given that DCs present antigens to T cells in lymph nodes to establish alloimmunity in organ transplantation, hepatic lymphangiogenesis might contribute to immune tolerance in the liver by sending tolerant hepatic DCs to lymph nodes.
      While lymphatics help to reduce inflammation, they also serve as routes of cancer metastasis.

      Congenital liver diseases

      Lymphoedema cholestasis syndrome (LCS) is an autosomal recessive syndrome characterised by primary lymphoedema and cholestasis in the neonatal period with intermittent recurrences in childhood. A Norwegian type of LCS (a.k.a., Aagenaes syndrome) has the same haplotype on chromosome 15q and is classified as LCS1.
      • Drivdal M.
      • Slagsvold C.E.
      • Aagenaes O.
      • Kase B.F.
      Hereditary lymphedema, characteristics, and variations in 17 adult patients with lymphedema cholestasis syndrome 1/Aagenaes syndrome.
      In a study of 40 patients with LCS1, 6 patients developed severe cirrhosis with death or liver transplantation in infancy or early childhood, and 3 patients slowly developed progressive cirrhosis.
      • Drivdal M.
      • Trydal T.
      • Hagve T.A.
      • Bergstad I.
      • Aagenaes O.
      Prognosis, with evaluation of general biochemistry, of liver disease in lymphoedema cholestasis syndrome 1 (LCS1/Aagenaes syndrome).
      Recently, a mutation of CCBE1, a secreted protein essential for VEGF-C activation, was reported to be responsible for LCS in a patient without the LCS1 mutation.
      • Shah S.
      • Conlin L.K.
      • Gomez L.
      • Aagenaes O.
      • Eiklid K.
      • Knisely A.S.
      • et al.
      CCBE1 mutation in two siblings, one manifesting lymphedema-cholestasis syndrome, and the other, fetal hydrops.
      In addition, the CCBE1 mutation was identified in another patient who developed recurrent cholangitis at age 52 with a family history of primary lymphoedema in lower limbs.
      • Viveiros A.
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      • Schaefer B.
      • Finkenstedt A.
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      • et al.
      CCBE1 mutation causing sclerosing cholangitis: expanding the spectrum of lymphedema-cholestasis syndrome.
      While the mechanism of cholestasis in LCS remains to be elucidated, these clinical findings may indicate that malfunctions of the hepatic lymphatic system are involved in its pathogenesis.

      NAFLD

      Increased lymphatic vessel density was observed in areas of fibrosis and immune cell infiltration in patients with chronic liver diseases, including NASH.
      • Tamburini B.A.J.
      • Finlon J.M.
      • Gillen A.E.
      • Kriss M.S.
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      • Fu R.
      • et al.
      Chronic liver disease in humans causes expansion and differentiation of liver lymphatic endothelial cells.
      In this study, single-cell RNA sequencing analysis of LECs from healthy controls and patients with NASH revealed upregulation of genes related to IL13 signalling,
      • Tamburini B.A.J.
      • Finlon J.M.
      • Gillen A.E.
      • Kriss M.S.
      • Riemondy K.A.
      • Fu R.
      • et al.
      Chronic liver disease in humans causes expansion and differentiation of liver lymphatic endothelial cells.
      which has been shown to regulate lymphangiogenesis negatively by inhibiting Prox1 expression as well as migration and proliferation of LECs.
      • Shin K.
      • Kataru R.P.
      • Park H.J.
      • Kwon B.I.
      • Kim T.W.
      • Hong Y.K.
      • et al.
      TH2 cells and their cytokines regulate formation and function of lymphatic vessels.
      ,
      • Savetsky I.L.
      • Ghanta S.
      • Gardenier J.C.
      • Torrisi J.S.
      • Garcia Nores G.D.
      • Hespe G.E.
      • et al.
      Th2 cytokines inhibit lymphangiogenesis.
      This study also showed, in a preclinical model of NASH, that oxidised low-density lipoprotein, which is known to be elevated in NASH livers,
      • Ampuero J.
      • Ranchal I.
      • Gallego-Durán R.
      • Pareja M.J.
      • Del Campo J.A.
      • Pastor-Ramírez H.
      • et al.
      Oxidized low-density lipoprotein antibodies/high-density lipoprotein cholesterol ratio is linked to advanced non-alcoholic fatty liver disease lean patients.
      ,
      • McGettigan B.
      • McMahan R.
      • Orlicky D.
      • Burchill M.
      • Danhorn T.
      • Francis P.
      • et al.
      Dietary lipids differentially shape nonalcoholic steatohepatitis progression and the transcriptome of kupffer cells and infiltrating macrophages.
      could induce IL13 upregulation and reduce Prox1 transcript levels and LEC identity.
      • Tamburini B.A.J.
      • Finlon J.M.
      • Gillen A.E.
      • Kriss M.S.
      • Riemondy K.A.
      • Fu R.
      • et al.
      Chronic liver disease in humans causes expansion and differentiation of liver lymphatic endothelial cells.
      Later, this group recapitulated these findings in mice fed a high-fat, high-cholesterol diet (another NASH model).
      • Burchill M.A.
      • Finlon J.M.
      • Goldberg A.R.
      • Gillen A.E.
      • Dahms P.A.
      • McMahan R.H.
      • et al.
      Oxidized low-density lipoprotein drives dysfunction of the liver lymphatic system.
      They also showed that lymphatic transport activity was impaired in NASH mice, but it was rescued by administration of recombinant vascular endothelial growth factor C (rVEGF-C) concomitant with amelioration of hepatic inflammation. Hence, drugs with pro-lymphangiogenic properties could be a new therapeutic strategy for NASH.
      In recent years, a growing body of evidence has indicated that impairment of lipid transport by the lymphatic system (i.e., lacteals) could have systemic metabolic consequences.
      • Blum K.S.
      • Karaman S.
      • Proulx S.T.
      • Ochsenbein A.M.
      • Luciani P.
      • Leroux J.C.
      • et al.
      Chronic high-fat diet impairs collecting lymphatic vessel function in mice.
      • Escobedo N.
      • Proulx S.T.
      • Karaman S.
      • Dillard M.E.
      • Johnson N.
      • Detmar M.
      • et al.
      Restoration of lymphatic function rescues obesity in Prox1-haploinsufficient mice.
      • Escobedo N.
      • Oliver G.
      The lymphatic vasculature: its role in adipose metabolism and obesity.
      • Harvey N.L.
      • Srinivasan R.S.
      • Dillard M.E.
      • Johnson N.C.
      • Witte M.H.
      • Boyd K.
      • et al.
      Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity.
      Similarly, many studies have addressed the relationship between lymphatic dysfunction and obesity. For example, in heterozygous mice lacking Prox1 (Prox1+/–), lymphatic vascular defects and adult-onset obesity with elevated triglycerides were observed.
      • Harvey N.L.
      • Srinivasan R.S.
      • Dillard M.E.
      • Johnson N.C.
      • Witte M.H.
      • Boyd K.
      • et al.
      Lymphatic vascular defects promoted by Prox1 haploinsufficiency cause adult-onset obesity.
      Lymphatic-specific Prox1 restoration rescued the adult-onset obesity phenotype in Prox1+/– mice, directly linking lymphatic dysfunction to the development of obesity. Obesity could also facilitate lymphatic dysfunction. Multiple mouse models of obesity demonstrated impaired lymphatic functions characterised by leaky lymphatics and reduced pumping capacities of collecting lymphatic vessels.
      • Blum K.S.
      • Karaman S.
      • Proulx S.T.
      • Ochsenbein A.M.
      • Luciani P.
      • Leroux J.C.
      • et al.
      Chronic high-fat diet impairs collecting lymphatic vessel function in mice.
      ,
      • García Nores G.D.
      • Cuzzone D.A.
      • Albano N.J.
      • Hespe G.E.
      • Kataru R.P.
      • Torrisi J.S.
      • et al.
      Obesity but not high-fat diet impairs lymphatic function.
      • Hespe G.E.
      • Kataru R.P.
      • Savetsky I.L.
      • García Nores G.D.
      • Torrisi J.S.
      • Nitti M.D.
      • et al.
      Exercise training improves obesity-related lymphatic dysfunction.
      • Nitti M.D.
      • Hespe G.E.
      • Kataru R.P.
      • García Nores G.D.
      • Savetsky I.L.
      • Torrisi J.S.
      • et al.
      Obesity-induced lymphatic dysfunction is reversible with weight loss.
      • Savetsky I.L.
      • Torrisi J.S.
      • Cuzzone D.A.
      • Ghanta S.
      • Albano N.J.
      • Gardenier J.C.
      • et al.
      Obesity increases inflammation and impairs lymphatic function in a mouse model of lymphedema.
      • Torrisi J.S.
      • Hespe G.E.
      • Cuzzone D.A.
      • Savetsky I.L.
      • Nitti M.D.
      • Gardenier J.C.
      • et al.
      Inhibition of inflammation and iNOS improves lymphatic function in obesity.
      Thus, it may be interesting to investigate NAFLD with a focus on impairment of lipid transfer due to lymphatic dysfunction.

      Hepatic encephalopathy

      Hepatic encephalopathy (HE) is a serious neurologic complication in patients with severe liver dysfunction resulting from acute liver failure or decompensated cirrhosis. In 2015, two groups of investigators re-discovered the presence of meningeal lymphatic vessels located underneath the skull
      • Aspelund A.
      • Antila S.
      • Proulx S.T.
      • Karlsen T.V.
      • Karaman S.
      • Detmar M.
      • et al.
      A dural lymphatic vascular system that drains brain interstitial fluid and macromolecules.
      ,
      • Louveau A.
      • Smirnov I.
      • Keyes T.J.
      • Eccles J.D.
      • Rouhani S.J.
      • Peske J.D.
      • et al.
      Structural and functional features of central nervous system lymphatic vessels.
      and revealed that they are the major route for discharging waste materials and immune cells from the brain to lymph nodes in the neck called the deep cervical lymph nodes. Because of its critical functions in brain homeostasis, the meningeal lymphatic system has been receiving a lot of attention as a potential therapeutic target for neurodegenerative and neuroinflammatory disorders.
      Modulation of lymphangiogenesis or lymphatic drainage could be an effective therapeutic strategy for a wide range of pathological conditions, including liver diseases.
      A recent study using rats with 4-week bile duct ligation provided insight into the role of the meningeal lymphatic system in HE.
      • Hsu S.J.
      • Zhang C.
      • Jeong J.
      • Lee S.I.
      • McConnell M.
      • Utsumi T.
      • et al.
      Enhanced meningeal lymphatic drainage ameliorates neuroinflammation and hepatic encephalopathy in cirrhotic rats.
      The study demonstrated that overexpression of VEGF-C via adeno-associated virus 8-VEGF-C injection to the cisterna magna ameliorated HE, including motor dysfunction, by decreasing neuroinflammation and microglia activation through increased meningeal lymphangiogenesis and thereby enhanced meningeal drainage to the cervical lymph nodes. Manipulation of meningeal lymphangiogenesis could be a novel therapeutic strategy for HE.

      Therapeutic potential and future directions

      An increasing body of evidence supports the idea that modulation of lymphangiogenesis or lymphatic drainage is an effective therapeutic strategy for a wide range of pathological conditions,
      • Oliver G.
      • Kipnis J.
      • Randolph G.J.
      • Harvey N.L.
      The lymphatic vasculature in the 21(st) century: novel functional roles in homeostasis and disease.
      ,
      • Schwager S.
      • Detmar M.
      Inflammation and lymphatic function.
      including rheumatoid arthritis,
      • Shi J.X.
      • Liang Q.Q.
      • Wang Y.J.
      • Mooney R.A.
      • Boyce B.F.
      • Xing L.
      Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice.
      psoriasis-like chronic dermatitis,
      • Schwager S.
      • Renner S.
      • Hemmerle T.
      • Karaman S.
      • Proulx S.T.
      • Fetz R.
      • et al.
      Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.
      inflammatory bowel disease,
      • D'Alessio S.
      • Correale C.
      • Tacconi C.
      • Gandelli A.
      • Pietrogrande G.
      • Vetrano S.
      • et al.
      VEGF-C-dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease.
      interstitial nephritis
      • Hasegawa S.
      • Nakano T.
      • Torisu K.
      • Tsuchimoto A.
      • Eriguchi M.
      • Haruyama N.
      • et al.
      Vascular endothelial growth factor-C ameliorates renal interstitial fibrosis through lymphangiogenesis in mouse unilateral ureteral obstruction.
      and glioblastoma.
      • Song E.
      • Mao T.
      • Dong H.
      • Boisserand L.S.B.
      • Antila S.
      • Bosenberg M.
      • et al.
      VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.
      For liver diseases, several preclinical studies have shown that administration of VEGF-C or D ameliorates disease conditions by increasing lymphangiogenesis and lymphatic drainage. On the other hand, blocking lymphangiogenesis could also be an appropriate option for diseases such as cholangiocarcinoma where tumour-induced lymphangiogenesis may promote metastasis of malignant tumours.
      An understanding of the detailed molecular and cellular mechanisms of hepatic lymphangiogenesis is essential for its modulation. It is not adequately known which cells produce pro- or anti-lymphangiogenic factors and regulate hepatic lymphangiogenesis. Macrophages have been shown to regulate hepatic lymphangiogenesis by producing lymphangiogenic factors such as VEGF-C and D at the site of lymphatic vessel expansion.
      • Nakamoto S.
      • Ito Y.
      • Nishizawa N.
      • Goto T.
      • Kojo K.
      • Kumamoto Y.
      • et al.
      Lymphangiogenesis and accumulation of reparative macrophages contribute to liver repair after hepatic ischemia-reperfusion injury.
      Cholangiocarcinoma cells produce PDGF-D, which induces VEGF-C expression in myofibroblasts in tumour microenvironments, facilitating lymphangiogenesis and subsequent metastasis.
      • Cadamuro M.
      • Brivio S.
      • Mertens J.
      • Vismara M.
      • Moncsek A.
      • Milani C.
      • et al.
      Platelet-derived growth factor-D enables liver myofibroblasts to promote tumor lymphangiogenesis in cholangiocarcinoma.
      These observations may indicate that distinct mechanisms of lymphangiogenesis exist in different liver diseases.
      While blocking VEGF-C is effective for decreasing lymphatic vessels and may prevent tumour metastasis, it will also reduce recruitment of T cells with potential tumour-killing abilities.
      • Song E.
      • Mao T.
      • Dong H.
      • Boisserand L.S.B.
      • Antila S.
      • Bosenberg M.
      • et al.
      VEGF-C-driven lymphatic drainage enables immunosurveillance of brain tumours.
      Thus, identification of alternative lymphangiogenic pathways other than the VEGF-C/D/VEGFR3 axis, including additional pro- and anti-lymphangiogenic factors, will likely increase novel therapeutic options for cholangiocarcinoma in particular and for liver diseases in general.
      • Tanaka M.
      • Iwakiri Y.
      The hepatic lymphatic vascular system: structure, function, markers, and lymphangiogenesis.
      A recent study showed that thrombospondin (THBS)1, THBS2 and pigment epithelial-derived factor released by ICC cells into the intrahepatic tumour microenvironment promoted cancer-associated lymhangiogenesis, suggesting that THBS1, THBS2 and pigment epithelial-derived factor could be promising targets to reduce cancer-associated lymphangiogenesis and counteract the invasiveness of ICC.
      • Carpino G.
      • Cardinale V.
      • Di Giamberardino A.
      • Overi D.
      • Donsante S.
      • Colasanti T.
      • et al.
      Thrombospondin 1 and 2 along with PEDF inhibit angiogenesis and promote lymphangiogenesis in intrahepatic cholangiocarcinoma.
      The origin of LECs responsible for hepatic lymphangiogenesis in pathological conditions remains to be identified. A previous study reported a very small contribution of bone marrow-derived hematopoietic stem cells to LECs in the regenerating mouse liver.
      • Jiang S.
      • Bailey A.S.
      • Goldman D.C.
      • Swain J.R.
      • Wong M.H.
      • Streeter P.R.
      • et al.
      Hematopoietic stem cells contribute to lymphatic endothelium.
      Further studies with lineage tracing analysis are warranted to identify the origin of LECs in hepatic lymphangiogenesis.
      Finally, it can be expected that hepatic lymph contains 80% to 90% of the proteins present in plasma[1, 3]. The content of the lymph, including self-peptides derived from intracellular, membrane-associated or matrix proteins, has been gaining attention as it may represent local conditions more accurately than blood,
      • Clement C.C.
      • Cannizzo E.S.
      • Nastke M.D.
      • Sahu R.
      • Olszewski W.
      • Miller N.E.
      • et al.
      An expanded self-antigen peptidome is carried by the human lymph as compared to the plasma.
      ,
      • Clement C.C.
      • Rotzschke O.
      • Santambrogio L.
      The lymph as a pool of self-antigens.
      and thus contain more useful biomarkers. Moreover, lymph carries apoptotic cellular materials, cytokines, cell-derived microparticles and infectious agents, mediating communications between lymph-generating organs (e.g., liver) and their associated draining lymph nodes, which is critical for host defence.
      • Clement C.C.
      • Rotzschke O.
      • Santambrogio L.
      The lymph as a pool of self-antigens.
      That said, the aetiological relevance of small molecules and vesicles released into the lymphatic system remains to be explored.
      In conclusion, the lymphatic system in the liver remains underexplored. Given its pivotal homeostatic roles in the liver and other organs, investigations in this area will advance our knowledge of liver physiology and pathophysiology and lead to the development of effective therapies for liver diseases.

      Abbreviations

      ADAMTS3, A distintegrin and metalloprotease with thrombospondin motifs-3; CAFs, cancer-associated fibroblasts; CCBE1, collagen and calcium binding EGF domains 1; CCl4, carbon tetrachloride; CCL21, C–C motif chemokine ligand 21; CCR7, C–C chemokine receptor type 7; DCs, dendritic cells; ECs, endothelial cells; HCC, hepatocellular carcinoma; HE, hepatic encephalopathy; ICAM1, intracellular adhesion molecule 1; ICC, intrahepatic cholangiocarcinoma; IL-7, interleukin-7; LCS, lymphedema cholestasis syndrome; LECs, lymphatic endothelial cells; LSEC, liver sinusoidal endothelial cell; Lyve1, lymphatic vessel endothelial hyaluronan receptor 1; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; PBC, primary biliary cholangitis; PDGF-D, platelet-derived growth factor D; Prox1, prospero homeobox protein 1; S1P, sphingosine-1-phosphate; S1P1, S1P receptor 1; THBS1, thrombospondin 1; VEGFR3, vascular endothelial growth factor receptor 3.

      Financial support

      This study was supported by NIH grants ( 1R56DK121511 , 1R01AA025342 , R01DK130362 and R01DK117597 ) to YI.

      Authors’ contributions

      All authors wrote and approved the final manuscript.

      Conflict of interest

      The authors declare no conflicts of interest that pertain to this work.
      Please refer to the accompanying ICMJE disclosure forms for further details.

      Acknowledgements

      We would like to thank Drs. Teruo Utsumi and Matthew McConnell (Yale University) for critical reading and editing.

      Supplementary data

      The following are the supplementary data to this article:

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