The Hepatic Lymphatic Vascular System: Structure, Function, Markers, and Lymphangiogenesis

Abstract

The lymphatic vascular system has been little explored in the liver despite its indispensable properties including maintenance of tissue fluid homeostasis. The discovery of specific markers for lymphatic endothelial cells has advanced various experimental methods including imaging, cell isolation and transgenic animal models, and has resulted in the rapid progress of the lymphatic vascular research in other tissues and organs during the last decade. These studies have also brought concrete evidence that lymphatic vessel dysfunction plays an important role in the pathogeneses of various diseases. This review will provide current knowledge of structure, function and markers of the hepatic lymphatic vascular system as well as factors associated with hepatic lymphangiogenesis in reference to those of other tissues.

Introduction

The lymphatic vascular system constitutes the major circulatory system together with the blood vascular system and is engaged in indispensable physiological activities. The lymphatic vascular system maintains tissue fluid homeostasis by collecting excess tissue fluid and returning it to the venous circulation. It also plays an essential role in absorption and transport of dietary fat. Further, lymphatic vessels are crucial for immune surveillance and acquired immunity by serving as the main conduits of antigens and antigen-presenting cells from peripheries to lymph nodes.^1–4

The lymphatic vascular research had been delayed partly because of lack of knowledge about markers and signaling pathways specific to the lymphatic vasculature. In 1995 to 1997, however, the important findings were brought, which showed that vascular endothelial growth factor receptor (VEGFR)-3 is expressed in the lymphatic endothelium and that its ligand vascular endothelial growth factor (VEGF)-C promotes lymphangiogenesis.^{5, 6} These findings on signaling pathways specific to the lymphatic vasculature and the subsequent discoveries of other specific markers for lymphatic endothelial cells (LyECs), such as lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1)⁷, prospero homeobox protein 1 (Prox1)⁸ and podoplanin⁹, have advanced the lymphatic vascular research. In consequence, it has been recognized that lymphatic vessel dysfunction plays an important role in the pathogeneses of various diseases.

In the liver, however, the study of the lymphatic vascular system has been little explored. This review will provide an overview of structure, function and markers of the lymphatic vascular system as well as factors associated with lymphangiogenesis in the liver along with knowledge gained in other tissues.

1. Structure of the hepatic lymphatic vascular system

This section will address structure of the lymphatic vascular system in general, followed by that of the liver. Detailed anatomical structure of the lymphatic and hepatic lymphatic vascular systems is available in other review articles.^{3, 10–12}

1) Anatomy of the Lymphatic Vascular System

Lymphatic capillaries

Lymphatic fluid originates from plasma components leaked from capillary vessels into the interstitium. Lymphatic fluid then enters into lymphatic capillaries, which are blind-ended, thin-walled vessels consisting of a single layer of LyECs. Lymphatic capillaries are not covered by pericytes or smooth muscle cells and lack basement membranes.^{13, 14} They are highly permeable with discontinuous “button-like” junctions, through which interstitial fluid, macromolecules and immune cells can be transported.¹⁵ In addition, LyECs have anchoring filaments that are mainly composed of emilin-1 and fibrillin and bind LyECs to the surrounding extracellular matrix.^{14, 16, 17} These filaments keep lymphatic vessel lumens open to facilitate tissue fluid intake in conditions of tissue swelling.

Collecting vessels

Lymphatic capillaries coalesce into collecting vessels, which are covered with smooth muscle cells and basement membranes.¹⁴ Collecting vessels are much less permeable than lymphatic capillaries because LyECs form “zipper-like” junctions. Collecting vessels can be divided into smaller functional units called lymphangions that have unidirectional bicuspid valves at each end.¹⁸ The phasic contraction of smooth muscle cells covering lymphangions has collecting vessels act as a pump to drive lymphatic flow. Stimulation of smooth muscle cells causes depolarization of cell membrane and opens Ca²⁺ channels, resulting in Ca²⁺ influx and smooth muscle cell contraction. Smooth muscle cells also have stretch-activated Ca²⁺ channels, facilitating phasic contraction.^{19, 20} On the other hand, LyECs produce nitric oxide (NO), a vasodilator, in response to shear stress by fluid flow, which counteracts the Ca²⁺-dependent contraction.^{21, 22} Therefore, spatiotemporal alterations of Ca²⁺ and NO levels are thought to modulate the phasic contraction of lymphangions.²³

Lymph nodes and lymph trunks

Collecting vessels are connected to one or more lymph nodes. Antigen presenting cells including dendritic cells and macrophages in lymphatic fluid interact with lymphocytes in lymph nodes, facilitating adaptive immune responses. Then, lymphatic fluid flows to secondary central lymph nodes, tertiary central lymph nodes and lymph trunks finally.²⁴ Lymphatic fluid from the left side of the body, abdomen and lower limb is ultimately drained into the thoracic duct, the largest lymphatic vessel, which is connected to the left subclavian vein (Figure 1). Lymphatic fluid from the other parts of the body is drained into the right lymph trunk connected to the right subclavian vein.²⁵ Lymphatic fluid entering into the subclavian veins returns to the systemic blood circulation.

Figure 1. The schematic diagram of the macro-anatomy of the hepatic lymphatic vascular system.

(➀): Lymphatic capillaries in the portal tract coalesce into collecting vessels, which drain to lymph nodes at the hepatic hilum and the lesser omentum. Efferent lymphatic vessels (LV) from these lymph nodes connect to celiac lymph nodes, which drain to the cisterna chyli, the enlarged origin of the thoracic duct. Lymphatic fluid through the thoracic duct drains to the left subclavicular vein and returns to the systemic blood circulation. (➁): Lymphatic vessels along the central vein (CV) converge into large lymphatic vessels along the hepatic vein (HV), which traverse along the inferior vena cava (IVC) through the diaphragm toward mediastinal lymph nodes. (➂): Lymphatic fluid running underneath the capsule of the liver-convex (3i) drains to mediastinal lymph nodes through the coronary ligament, while that of the liver-concave surface (3ii) drains to lymph nodes of the hepatic hilum and regional lymph nodes. HA, hepatic artery; PV, portal vein; BD, bile duct; LN, lymph node.

2) Anatomy of the Hepatic Lymphatic Vascular System

The schematic diagram of the hepatic lymphatic vascular system is shown in Figures 1 and 2. The liver has sinusoids instead of capillaries.²⁶ Sinusoids are distinct from capillaries as they consist of one layer of liver sinusoidal endothelial cells (LSECs) and lack pericytes and basement membranes, similar to lymphatic capillaries. The origin of hepatic lymphatic fluid is thought to be plasma components filtered through fenestrae of LSECs into the space of Disse, the interstitial space between LSECs and hepatocytes.^{10, 11} Lymphatic fluid in the space of Disse mostly flows through the space of Mall, a space between the stroma of the portal tract and the outermost hepatocytes²⁷, into the interstitium of the portal tract and then into lymphatic capillaries. Some portion of the lymphatic fluid in the space of Disse flows into the interstitium around the central vein, which is located in the center of the liver acinus and connected to the hepatic vein²⁸, or underneath the hepatic capsule (Figure 2).

Figure 2. The schematic diagram of the micro-anatomy of the hepatic lymphatic vascular system.

Blood flow (red arrows) from the portal vein (PV) and the hepatic artery (HA) enters to the liver. Plasma components filtered through liver sinusoidal endothelial cells (LSECs) into the space of Disse, the interstitial space between LSECs and hepatocytes, are regarded as the origin of lymphatic fluid. Lymphatic fluid in the space of Disse mostly flows through the space of Mall, the space between the stroma of the portal tract and the outermost hepatocytes, into the interstitium of the portal tract and then into lymphatic capillaries (➀). Some portion of the lymphatic fluid in the space of Disse flows into the interstitium around the central vein (➁) or underneath the hepatic capsule (➂).

Lymphatic capillaries in the portal tract coalesce into collecting vessels and drain to lymph nodes at the hepatic hilum. Lymphatic vessels along the central vein converge into 5–6 large lymphatic vessels, which traverse along the inferior vena cava through the diaphragm toward posterior mediastinal lymph nodes. Lymphatic fluid running underneath the capsule of the liver-convex drains to mediastinal lymph nodes through the coronary ligament, while that of the liver-concave surface drains to lymph nodes of the hepatic hilum and regional lymph nodes (Figure 1).^{10–12, 29} Based on their locations, lymphatic vessels along the portal tract and the central vein are called the deep lymphatic system and those along the hepatic capsule are called the superficial lymphatic system.^{10–12, 29}

2. Markers of lymphatic vessels

Lymphatic vessel markers generally refer to those of LyECs, reflecting their structural significance. Among those markers, LYVE-1^{7, 30, 31}, podoplanin^{9, 32}, Prox1^{8, 33–35} and VEGFR-3⁵ are most commonly used for microscopic imaging of lymphatic vessels.³⁶ Finding more specific markers for the liver may be needed, because most well known LyEC markers, such as LYVE-1 and Prox1, are also expressed in liver sinusoidal endothelial cells (LSECs) and hepatocytes, respectively. Table 1 summarizes LyEC markers whose expressions were histologically examined in the liver. Some of them are also briefly discussed below.

Table 1.

Lymphatic markers

Marker	Postnatal expression EXCEPT for lymphatic vessels		Hepatic expression in pathological conditions	Ref.
	Liver	Other organs/cells
LYVE-1	Sinusoidal endothelial cells	A portion of macrophages, pulmonary capillaries, epididymal adipose tissue, mesentery, eye (cornea, sclera, choroid, iris, and retina), wounded skin, and malignant tumors (melanoma and insulinoma)	In chronic hepatitis and liver cirrhosis in humans, LYVE-1(+) lymphatic vessels increase but LYVE-1(+) sinusoidal endothelial cells decrease.	38, 40–43, 107, 161–166
Prox1	Hepatocytes	Adrenal medulla, megakaryocytes, and platelets	Intra-hepatic CCC, ductular cells in cirrhotic livers, and HCC in humans.	8, 52, 59, 60
Podoplanin	Cholangiocytes	Inflammatory macrophages, mesothelial cells, cardiomyocytes, FRCs, follicular dendritic cells, TH17 cells, and osteoblasts	Podoplanin(+) lymphatic vessels increase in decompensated cirrhosis in humans. Podoplanin(+) FRCs increase in the livers of primary biliary cirrhosis patients. EHE and angiomyolipoma in humans.	73, 74, 77–81, 167, 168
VEGFR-3	Cholangiocytes	A portion of macrophages, proliferating blood vessels, and fenestrated capillaries in endocrine glands, choroid plexus, kidney, and small intestine	HBx Ag positive HCC and hepatic progenitor cells in primary biliary cirrhosis in humans.	82, 85–87, 104, 169, 170
CCL21	Sinusoidal endothelial cells	A portion of dendritic cells, HEVs of lymph nodes and Peyer’s patches, T-cell areas of spleen, lymph nodes, and Peyer’s patches	Lymphoid tissue in primary biliary cirrhosis and primary sclerosing cholangitis in humans.	171–173
MMR1	Sinusoidal endothelial cells and Kupffer cells	A portion of macrophages, sinusoidal endothelial cells in bone marrow and spleen, perivascular microglia, and glomerular mesangial cells	Unknown	174–177
Desmoplakin	Basolateral plasma membrane of hepatocytes and cholangiocytes	Esophagus, intestine, colon, salivary gland, mammary gland, sweat gland, thymus, and endocervix	Entire plasma membrane of HCC cells	176, 178–181
Integrin α9	Hepatocytes	Airway epithelial cells, keratinocytes, muscle cells (smooth/skeletal/cardiac), neutrophils, osteoclasts, and oocytes	Unknown	176, 182

[Abbreviations] LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; Prox1, prospero homeobox protein 1; CCC, cholangiocellular carcinoma; HCC, hepatocellular carcinoma; FRC, fibroblastic reticular cell; EHE, epithelioid hemangioendothelioma; VEGFR, vascular endothelial growth factor receptor; HBx Ag, Hepatitis B x antigen; CCL21, C-C motif chemokine ligand 21; HEV, high endothelial venules; MMR, macrophage mannose receptor 1

LYVE-1

LYVE-1 is a lymphatic vessel endothelial hyaluronan (HA) receptor, which belongs to the Link protein superfamily that contains a conserved HA-binding domain, known as the Link module.³⁷ LYVE-1 is a homolog of the CD44 HA receptor.⁷ With a Link module and homology shared with the CD44 HA receptor, LYVE-1 may be involved in the transport of HA across the lymphatic endothelium. LYVE-1 is strongly expressed on the entire luminal and abluminal surfaces of LyECs, even on fine filopodia of growing vessels during lymphangiogenesis.

No definite alterations in lymphatic vessel structure and function were reported for LYVE-1^−/− mice.³⁸ However, diphtheria toxin-induced LYVE-1 depletion in mice caused acute loss of lymphatic lacteals in intestinal villi and lymphatic vessels in systemic lymph nodes. These changes resulted in the structural distortion of blood capillaries and whole architecture of the villi, leading to death due to sepsis within 60 hours after LYVE-1 depletion.³⁹ These observations indicate that LYVE-1 plays an important role in the maintenance of the lymphatic vascular system, especially lacteals in intestinal villi and lymph nodes. Therefore, some compensatory mechanisms might work in the setting of congenital loss of LYVE-1.

In the liver, LYVE-1 is expressed not only in LyECs but also in LSECs in mice⁴⁰ and humans.^40–43 However, LYVE-1 positivity in LSECs was reported to diminish in inflamed human livers, such as those of chronic hepatitis and cirrhosis.^{40, 42} Expression levels of LYVE-1 in human hepatocellular carcinoma (HCC) negatively correlated with the overall survival of patients.⁴⁴

Prox1

Prox1, a homolog of the Drosophila melanogaster homeobox gene prospero, is a transcriptional factor and regulates the genes related to lymphatic endothelial cells, such as VEGFR-3 and podoplanin.^{45, 46} Prox1 is essential for the development of the lymphatic vascular system,⁸ while it also plays a role in the development of other tissues, including the lens^{34, 47}, retina⁴⁸, heart⁴⁹, central nervous system⁵⁰, pancreas⁵¹ and liver^{52, 53}. Prox1 is positive in the nucleus in contrast to other lymphatic markers that are expressed in the cytoplasm and/or the plasma membrane.

Prox1^−/− mice are devoid of the lymphatic vascular system and die at approximately E14.5 because Prox1 is essential for budding of lymphatic endothelial sacs.⁸ Prox1^+/− mice die in a few days after their birth and present dysfunction of lymphatic vessels with chylous ascites.^{8, 47, 54} Several lines of Prox1-promoter directed reporter mice have recently been established as effective research tools (GFP⁵⁵, mOrange⁵⁶ and tdTomato^57–59).

In early endoderm, Prox1 expression is restricted to the primordia of the liver and the pancreas.⁵¹ Prox1 regulates hepatocyte migration during liver morphogenesis⁵¹ and is expressed in postnatal hepatocytes, but not in postnatal pancreas.⁵² In humans, cholangiocytes of normal livers were negative for Prox1, but intra-hepatic cholangiocarcinoma and ductular cells in fibrotic septa of cirrhotic livers and HCC were positive.⁶⁰ In addition, expression levels of Prox1 in human HCC negatively correlated with the overall survival of patients.⁶¹ Prox1 acts with nuclear receptors, such as hepatocyte nuclear factor (HNF) 4α⁶², estrogen-related receptor (ERR) α^{63, 64}, liver receptor homolog (LRH)-1⁶⁵ and retinoic acid-related orphan receptors (ROR) α/γ⁶⁶, and regulates bile acid synthesis⁶⁵ and circadian metabolism in the liver.^{64, 66}

Podoplanin

Podoplanin is a type I transmembrane glycoprotein and is known to be essential for the development of the heart^67–70, lung⁷¹, spleen and lymph nodes⁷². Its expression is regulated by Prox1.⁴⁵ Podoplanin is also a ligand of C-type lectin receptor CLEC-2, which is highly expressed in platelets and immune cells and promotes platelet aggregation and activation.⁷³

Podoplanin^−/− mice die at their birth due to respiratory failure. These mice present congenital lymphedema due to lymphatic vessel defect although blood vessel formation is normal.⁷⁴ Podoplanin^+/− mice are healthy and fertile only with partial incompleteness of the lymphatic vessel network.⁷⁴ Recently, keratinocyte-specific podoplanin deficient mice⁷⁵ and a tamoxifen-inducible podoplanin depletion mouse model (Pdpn^f/f; CagCre)⁷⁶ have been developed.

Histological analysis of normal mouse livers showed expression of podoplanin in cholangiocytes besides LyECs.⁷⁷ Podoplanin-positive lymphatic vessels increased in human livers of decompensated cirrhosis.⁷⁸ It was also reported that podoplanin-positive fibroblastic reticular cells increased in human livers of primary biliary cirrhosis.⁷⁹ Podoplanin is a useful histological marker for diagnosing patients with vascular tumors with lymphatic differentiation, such as epithelioid hemangioendothelioma (EHE)⁸⁰ and angiomyolipoma.⁸¹

VEGFR-3

VEGFR-3 is a membrane-anchored tyrosine kinase and the receptor of VEGF-C and VEGF-D. VEGFR-3 plays a crucial role in lymphangiogenesis. In early embryogenesis prior to LyEC differentiation, VEGFR-3 is expressed in most endothelial cells, but in the later stages of development, its expression becomes mostly restricted to the lymphatic endothelium.⁵

VEGFR-3^−/− mice present lymphatic vessel defect and die at approximately E10.5.⁸² VEGFR-3^+/− mice present leaky lymphatic vessels and transient chylous ascites.^{82, 83} A mouse line (Vegfr3^EGFPLuc), in which a dual reporter for fluorescence and luminescence is expressed under VEGFR-3-promoter, was established recently and luminescence imaging of tumor-induced lymphangiogenesis became available.⁸⁴

VEGFR-3 expression was seen in cholangiocytes of normal rat livers and increased in those of cholestatic rat livers induced by bile duct ligation.⁸⁵ Hepatic progenitor cells were also found to express VEGFR-3 in patients with primary biliary cirrhosis.⁸⁶ Hepatitis B x antigen (HBx Ag) is one of the antigens of hepatitis B virus (HBV) and promotes hepatocarcinogenesis by upregulating expression of genes associated with proliferation of hepatocytes. Upregulation of VEGFR-3 expression was observed in HBx Ag-positive human HCC, and the prognosis of patients with VEGFR-3 positive HCC was worse than that with VEGFR-3 negative HCC.⁸⁷

3. Lymphangiogenesis

This section addresses the mechanism of lymphangiogenesis in the postnatal stage and factors that affect lymphangiogenesis, including inflammatory cells, in the lymphatic system in general. Then, implications of lymphangiogenesis in the pathophysiology of the liver are summarized.

1) Factors associated with lymphangiogenesis

In the postnatal stage, lymphatic vessels are mostly quiescent. Therefore, lymphangiogenesis generally occurs in pathological conditions such as tissue repair, inflammation and tumor-related conditions.⁸⁸ Many cytokines/growth factors have been reported to promote lymphangiogenesis (lymphangiogenic) or inhibit lymphangiogenesis (anti-lymphangiogenic), which are summarized in Table 2. The extent and duration of lymphangiogenesis are determined by balances between lymphangiogenic factors and antilymphangiogenic factors.^{89, 90}

Table 2.

Lymphangiogenic and Anti-lymphangiogenic Factors

Lymphangiogenic factors	Experimental model	Remarks	Ref.
VEGF-A	Mouse corneal lymphangiogenesis	VEGF-A recruits macrophages, which promote lymphangiogenesis by secreting VEGF-C/VEGF-D.	105
	Mouse subcutaneous immunization model	VEGF-A expression is upregulated concomitantly with lymphangiogenesis in LNs of immunized mice.	119
	Oxazolone sensitized delayed-type hypersensitivity in mouse ear	Systemic blockade of VEGF-A attenuates lymphangiogenesis in draining LNs.	183
	HSV-1 infection of cornea	HSV-1 causes lymphangiogenesis by promoting infected cells to secrete VEGF-A.	184
VEGF-C, VEGF-D	VEGF-C transgenic mouse	VEGF-C promotes LyEC proliferation and LV enlargement in the skin.	6
	Isolated LyEC	VEGF-C stimulates survival, growth, and migration of LyEC.	93
	FGF-2-induced corneal lymphangiogenesis	VEGFR-3 blockade cancels lymphangiogenesis.	185
	Chronic airway inflammation	VEGFR-3 blockade cancels lymphangiogenesis.	186
	LPS-induced peritonitis	VEGF-C and VEGF-D promote lymphangiogenesis in diaphragm.	187
Ang 2	Mouse corneal lymphangiogenesis	Ang 2 is upregulated in inflamed cornea and Ang2 blockade inhibits inflammatory lymphangiogenesis.	188
	Mouse corneal lymphangiogenesis	Ang 2 is expressed in lymphatic vessels and macrophages in inflamed cornea. Inflammatory lymphangiogenesis of cornea is suppressed in Ang2 knockout mice. Ang2 blockade inhibits LyEC proliferation and capillary tube formation.	189
HGF	Canine primary LyEC, rat tail lymphedema	HGF promotes proliferation and migration of LyEC. Weekly HGF gene transfer improves lymphedema in vivo.	190
LT	CCL21 transgenic mouse, RAG knockout mouse defective in T and B cell	LT overexpression by CCL21 transgene promotes lymphangiogenesis in thyroid. T cell depletion cancels this phenomenon.	191
	LTα knockout mouse, LTα transgenic mouse	LTα gene deletion decreases LV. Ectopic LTα expression causes lymphangiogenesis in tertiary lymphoid organs.	192
IL-1β	Mouse corneal lymphangiogenesis	IL-1β promotes lymphangiogenesis by upregulating expression of VEGF-A, VEGFC, and VEGF-D.	193
IL-7	Breast cancer cell lines, subcutaneous injection of MatrigelTM and/or IL-7 and/or breast cancer cell lines	IL-7 promotes VEGF-D expression of cell lines in vitro and promotes lymphangiogenesis in vivo.	194
	HECV cell line (originated from human umbilical cord), subcutaneous injection of MatrigelTM and/or IL-7 and/or HECV cell	IL-7 promotes expression of Prox1, LYVE-1 and podoplanin and proliferation, migration and tubular formation of LyEC via upregulation of VEGF-D.	195
IL-8	Human primary LyEC, IL-8 transgenic mouse and Prox1-GFP mouse	IL-8 promotes proliferation, migration and tube formation of LyEC. IL-8 overexpression promotes lymphangiogenesis in vivo.	196
IL-17	Cornea micro pocket assay, autoimmune ocular disease mouse	IL-17 promotes proliferation of LyEC via upregulation of VEGF-D. Blockade of IL-17 decreases corneal lymphangiogenesis.	197
IL-20	Human telomerase-transfected dermal LyEC	IL-20 promotes proliferation, migration and tubular formation of LyEC via PI3K and mTOR pathways.	198
Anti-lymphangiogenic factors	Experimental model	Remarks	Ref.
TGF-β	Human dermal lymphatic microvascular endothelial cells	TGF-β inhibits LyEC proliferation, cord formation, migration, expression of lymphatic markers (LYVE-1, Prox1) and lymphangiogenesis by VEGF-A/C via TGF-β type I receptor.	199
	Mouse tail skin excision and lymphatic vessel ligation	TGF-β1 inhibition promotes lymphatic vessel regeneration. TGF-β1 inhibits LyEC proliferation and fibrosis.	200
	Biopsy specimens from limbs of secondary lymphedema patients and mouse tail skin excision	TGF-β1 positive cells increase threefold in human lymphedema specimens. TGFβ1 inhibition decreases fibrosis, increases lymphangiogenesis and lymphatic function.	201
BMP2	Zebrafish BMP2 transgenic model	BMP2 inhibits LyEC differentiation from cardinal veins via inhibition of Prox1 expression.	202
IFN-α, IFN-γ	LyEC isolated from pig thoracic duct	IFN-α or IFN-γ decreases LyEC proliferation and migration. Treatment with both IFN-α and IFN-γ promotes LyEC apoptosis.	203
	Cervical LNs of T-cell deprived mouse	T cells inhibit lymphangiogenesis in LNs by secreting IFN-γ.	118
IL-4, IL-13	Mouse LyEC isolated from LNs, human dermal LyEC, mouse asthma model	IL-4 and IL-13 inhibit expression of Prox1 and LYVE-1 and tube formation of LyEC. Blockade of IL-4 and/or IL-13 increases the density and function of lung LVs in asthma model.	204
IL-27	Human dermal lymphatic microvascular endothelial cells	IL-27 inhibits LyEC proliferation and migration via STAT1/CXCL10, CXCL-11 axis.	205
Activin A	Subcutaneous injection of melanoma cell line to mouse	Activin A reduces lymphangiogenesis in melanoma model and inhibits sprouting of LyEC via phosphorylation of SMAD2.	206

[Abbreviations] VEGF, vascular endothelial growth factor; LN, lymph node; HSV-1, Herpes simplex virus 1; LyEC, lymphatic endothelial cell; LV, lymphatic vessel; FGF-2, fibroblast growth factors-2; VEGFR, vascular endothelial growth factor receptor; LPS, lipopolysaccharide; HGF, hepatocyte growth factor; LT, lymphotoxin; IL, interleukin; Prox1, prospero homeobox protein 1; LYVE-1, lymphatic vessel endothelial hyaluronan receptor 1; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; mTOR, mammalian target of rapamycin; TGF, tumor growth factor; IFN, interferon; STAT, signal transducer and activator of transcription; Ref, references.

2) Intracellular signaling pathways in lymphangiogenesis

Various growth factors activate their receptors on the surface of LyECs and initiate diverse signaling cascades that lead to the growth of lymphatic vessels (Table 2). These signaling pathways have largely been determined in studies of developmental lymphangiogenesis. Signaling via VEGF-C/D and VEGFR-3 is the most well-known pathway for lymphangiogenesis (Figure 3).⁶ VEGF-C or VEGF-D binding to VEGFR-3 results in autophosphorylation of multiple C-terminal tyrosine residues in VEGFR-3⁹¹, which transduces signaling through the Ras/Raf/MEK/ERK pathway.⁹² Signal transduction also occurs through the PI3K/Akt pathway⁹³ which causes phosphorylation of Akt, thereby activating mTOR and Rac1.⁹⁴ Activation of these signaling pathways facilitates LyEC proliferation and migration, i.e. lymphangiogenesis.⁹³ As discussed later, chronic inflammation and malignant tumors in the liver are known to induce several pro-lymphangiogenic growth factors including VEGF-C/D. However, a direct link between these increased pro-lymphangiogenic growth factors and lymphangiogenesis in these pathological conditions remains to be demonstrated (Figure 3). Excellent review papers are available for detailed signaling pathways in lymphangiogenesis.^95–97

Figure 3. Intracellular signaling pathways in lymphangiogenesis.

Signaling via VEGF-C/D and VEGFR-3 is the most well-known pathway for lymphangiogenesis. VEGF-C or VEGF-D binds to its receptor VEGFR-3 in the plasma membrane of lymphatic endothelial cells (LyECs), which facilitates signal transduction through various intracellular signaling pathways, leading to lymphangiogenesis. In the liver, activated macrophages in chronic inflammatory conditions, such as chronic hepatitis and liver cirrhosis, secrete VEGF-C and/or VEGF-D. Hepatic malignant tumors, such as hepatocellular carcinoma and intrahepatic cholangiocarcinoma, also secrete VEGF-C and/or VEGF-D. Further, these malignant tumors activate tumor-associated macrophages, which also secrete VEGF-C and/or VEGF-D. These secreted VEGF-C and VEGF-D are likely related to lymphangiogenesis in liver diseases through the VEGFR-3 mediated pathways.

3) Role of immune cells

Adaptive immune responses are initiated by migration of immune cells to inflamed sites. These immune cells phagocytose pathogens and transmigrate through lymphatic vessels to lymph nodes to present antigens to T cells. However, immune cells not only migrate through lymphatic vessels but also interact with lymphatic vessels and promote lymphangiogenesis.⁹⁸ An increase in lymphatic vessels helps infiltrating immune cells to evacuate from inflamed sites via lymphatic vessels and accelerates resolution of inflammation.^99–101

Macrophage

Among various immune cells, macrophages most highly interact with lymphatic vessels. LyECs secrete chemotactic factors, such as C10, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1), to attract macrophages.¹⁰² Macrophages secrete lymphangiogenic cytokines, such as VEGF-C, VEGF-D and VEGF-A¹⁰³, and promote tumor-associated lymphangiogenesis¹⁰⁴ and inflammation-induced lymphangiogenesis in the cornea¹⁰⁵, skin¹⁰⁰ and tail¹⁰⁶. Macrophages were recently indicated to have the ability to transdifferentiate to LyECs^107–109. However, this phenomenon is still controversial and needs further investigations.

Dendritic cell

Upregulation of inflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1β in inflamed tissues promotes expression of chemokines (e.g., CCL21/CCL19 and CXCL12) and their receptors (e.g., CCR7 and CXCR-4) in LyECs and dendritic cells^110–113, which enhances transmigration of dendritic cells through LyECs.^{114, 115} Inflammatory cytokines also increase expression of adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and E-selectin in LyECs and promote dendritic cell transmigration to lymphatic vessels.¹¹⁶ Dendritic cells were also reported to secrete VEGF-C and promote lymphangiogenesis.¹¹⁷

T cell

In a mouse model of tail lymphedema, nude mice exhibited less edema than wild-type mice concomitant with decreased lymphangiogenic cytokines and increased antilymphangiogenic cytokines. The balance of these cytokines was modulated by T-cell mediated inflammation.⁹⁰ T cells negatively regulated lymph node lymphangiogenesis by secreting interferon (IFN)-γ in mice.¹¹⁸

B cell

B cells promoted lymphangiogenesis in inflamed lymph nodes by secreting a robust amount of VEGF-A in mice given keyhole limpet hemocyanin (KLH) emulsified in complete Freund’s adjuvant (CFA) (an experimental model of inflamed lymph nodes).¹¹⁹ Interestingly, VEGF-C was not detected in this study. Another study using transgenic mice overexpressing VEGF-A specifically in B cells showed increased lymphangiogenesis as well as angiogenesis.¹²⁰

Neutrophil

Neutrophils are reported to contribute to lymphangiogenesis by modulating bioavailability and bioactivity of VEGF-A and secreting VEGF-D.¹²¹ Bioavailability of VEGF-A is increased by the secretion of matrix metalloproteinases 9 and heparanase. Depletion of neutrophils in mice developed skin inflammation in response to immunization or contact hypersensitization. Further, lymphangiogenesis was decreased in these mice with increased local inflammation, suggesting that neutrophils play a role in lymphangiogenesis and that lymphangiogenesis is helpful for reducing inflammation.

4) Lymphangiogenesis in the liver

Given that 25–50% of lymph passing through the thoracic duct originates from the liver^1,122, the liver can be considered the most important organ for lymphatic fluid production. However, the lymphatic vascular system in the liver has not been much explored. Only several studies have reported the occurrence of hepatic lymphangiogenesis in pathological conditions such as chronic hepatitis, liver fibrosis/cirrhosis, portal hypertension, malignant tumors and post-transplantation. This section summarizes these studies.

Chronic hepatitis, liver fibrosis and cirrhosis

The resistance to the blood flow in sinusoids increases in cirrhotic livers due to deformation of architecture including portal and central venules. Consequently, the hydrostatic pressure of sinusoids is elevated and plasma components filtrated through sinusoids, i.e., lymphatic fluid, increase. In cirrhotic patients, lymphatic fluid produced in the liver increased up to 30-fold^123–127 and peritoneoscopic observation showed dilation of lymphatic vessels in the liver surface.¹²⁸

Ascites formation in association with cirrhosis is one of the most recognized clinical manifestations of lymphatic vascular system disorders. How ascites is formed still remains to be elucidated. While several theories have developed^129–131, currently the most accepted one is “the peripheral arterial vasodilation theory or “the forward theory”.^132–134 According to this theory, splanchnic arterial vasodilation caused by portal hypertension results in underfilling of the splanchnic arterial circulation or hypovolemia. In moderate stages, the hypovolemia is compensated by renal retention of sodium and water. However, severe portal hypertension and splanchnic arterial vasodilation make sodium and water retention persistent and lead to leakage of fluid into the peritoneal cavity. When its amount exceeds the absorption capacity of lymphatic vessels, ascites results.^{131, 135}

On a related note, impaired lymphatic drainage in the splanchnic and peripheral regions was reported in cirrhotic rats with ascites. This was accompanied with a significantly increased activity of eNOS and increased production of nitric oxide (NO) in LyECs of these regions.¹³⁶ In addition, smooth muscle cell coverage of the lymphatic vessels in these regions was significantly decreased. Treatment of these cirrhotic rats with a NOS inhibitor significantly improved lymphatic drainage, decreased ascites volume and increased smooth muscle cell coverage. This study thus suggests the role of NO in impairment of lymphatic vessels in the splanchnic and peripheral regions and the development of ascites. It is not known whether lymphatic vessels in cirrhotic livers show similar pathological features.

The occurrence of hepatic lymphangiogenesis was reported for the first time in liver fibrosis/cirrhosis by Vollmar B. et al. in 1997.¹³⁷ They found lymphatic vessels increased and enlarged in cirrhotic rat livers induced by carbon tetrachloride (CCl₄). These observations of hepatic lymphatic vessels were confirmed in the following year in patients with chronic viral hepatitis/cirrhosis.¹³⁸

Microarray analysis demonstrated a 4-fold increase in VEGF-D expression in endothelial cells from CCl₄-induced cirrhotic rat livers, compared to control rat livers. Given that VEGF-D is a well-known lymphangiogenic factor with binding to VEGFR-3¹³⁹, which was also highly expressed in LyECs of these cirrhotic rats⁵, increased VEGF-D could be associated with lymphangiogenesis observed in liver cirrhosis (Figure 3).

Lymphangiogenesis was also reported to occur in idiopathic portal hypertension in human patients.¹⁴⁰ It was presumed that increased lymph production due to increased portal pressure caused lymphangiogenesis. In two rat models of portal hypertension (portacaval shunt and portal vein ligation), upregulation of Vegfr-3 expression was observed, leading us to speculate the occurrence of lymphangiogenesis.¹⁴¹ In any case including those of chronic hepatitis and liver fibrosis/cirrhosis mentioned above, the significance and the mechanism of hepatic lymphangiogenesis remain unknown.

Malignant tumors

Lymphatic vessels play a pivotal role in the pathogenesis of malignant tumors by serving as a pathway through which tumor cells spread from their original places to other places. The incidence of lymph node metastasis differs among tumors. For example, it is 5.1% in hepatocellular carcinoma and is 45.1% in intrahepatic cholangiocarcinoma. The prognosis of tumor-bearing patients with lymph node metastasis is worse than the cases without metastasis.^{142, 143} Many malignant tumors are known to secrete lymphangiogenic factors such as VEGF-C and VEGF-D and promote lymphangiogenesis in their adjacent tissues, which helps tumor cells to metastasize to lymph nodes.¹⁴⁴ In particular, many studies have demonstrated that tumor-associated macrophages play a vital role in lymphangiogenesis in malignant tumors by secreting VEGF-C and VEGF-D.^{104, 145–147} In intrahepatic cholangiocarcinoma, the lymphatic vessel density of surgically resected tumors was positively correlated with the incidence of lymphatic metastasis.¹⁴⁸ In hepatocellular carcinoma, VEGF-C expression was positively correlated with the size of tumors and the number of extrahepatic metastasis, and was negatively correlated with disease-free survival time.¹⁴⁹ Thus, blockade of VEGF-C may be a potential therapeutic strategy against malignant tumors. In fact, VEGF-C neutralizing antibody (VGX-100) is under a Phase I clinical trial for adult patients with advanced or metastatic solid tumors (NCT01514123).¹⁵⁰

Post-transplant lymphangiogenesis

In solid organ transplantations, the connection of lymphatic vessels between the graft and the recipient is interrupted at the time of operation. Since lymphatic vessels are essential for adaptive immunity, the association between lymphangiogenesis and graft rejection has received attention. Post-transplant lymphangiogenesis in grafts was associated with acute cellular graft rejection in various human transplantations (kidney^151–153, heart¹⁵⁴ and lung¹⁵⁵). However, a question still remains in regard to the pathological role of post-transplant lymphangiogenesis in graft rejection.¹⁵³ Post-transplant lymphangiogenesis could be detrimental if newly formed lymphatic vessels promote antigen presentation in draining lymph nodes and provoke alloimmune responses that result in graft rejection. However, it could be beneficial if these newly formed lymphatic vessels efficiently clear immune cells. In fact, in a rat model of liver transplantation, post-transplant lymphangiogenesis in grafts was associated with a long-term survival of recipients for more than 90 days.¹⁵⁶ In addition, rats that failed grafting by 11 days with acute cellular rejection and antibody-mediated rejection showed disappearance of lymphatic vessels from severely rejected areas, suggesting that lymphatic vessels take an important part in mitigation of inflammation at least in the early stage of transplantation. Further investigations to determine the mechanism and the time course of clearance of infiltrating immune cells by lymphatic vessels, especially in the early period of post-transplantation, may help successful grafting.

Conclusion and perspective

The lymphatic vascular system has been poorly studied in the liver. To drive research in this area, it is essential to identify better LyEC markers that do not overlap with LSECs, hepatocytes and other liver cells. Further, the development of experimental models for studying the lymphatic vascular system in postnatal livers will help to examine its role and molecular mechanisms in physiological and pathophysiological conditions. While all subjects are virtually novel in this area, it may be helpful to raise some specific questions to initiate the study.

First, the mechanism of hepatic lymphangiogenesis is largely unknown. The VEGFC/VEGFR-3 axis is considered the most potent signaling pathway that regulates lymphangiogenesis.⁹⁷ However, cellular sources of VEGF-C and VEGFR-3 have not been fully identified in the liver. Further, as shown in Table 2, many other molecules are reported to regulate lymphangiogenesis. These molecules are mostly observed in the liver in physiological and pathophysiological conditions. It is worth characterizing these molecules in relation to hepatic lymphangiogenesis.

Second, the relation between the lymphatic vascular system and metastasis is well known. The growth of lymphatic capillaries in liver tumors has been observed. However, the role of their growth in the development and the progression of liver tumors is largely undetermined. Like angiogenesis, it would be interesting to investigate lymphangiogenesis in liver cancer.

Third, inflammation is closely related to the development of many liver diseases. Infiltrating immune cells are drained to lymphatic vessels. Thus, it would be interesting to examine lymphangiogenesis in relation to inflammation. It is also unknown how immune cells can recognize lymphatic vessels to migrate. Elucidation of these mechanisms may help to develop anti-inflammatory strategies by facilitating immune cell clearance.

Fourth, on a biological matter, although LyECs are derived from cardinal veins^{8, 83} and LSECs are derived from the septum transversum¹⁵⁷, LyECs and LSECs have many similarities. As described previously, both LyECs and LSECs express LYVE-1.^40–43 VAP-1, a type II transmembrane protein that supports leukocyte adhesion, and Reelin, a glycoprotein that is associated with embryonic development, are also expressed in LyECs and LSECs.^{158, 159} In normal conditions, both LyECs and LSECs do not have basement membranes in lymphatic capillaries and sinusoids, respectively. Examining similarities and differences of these two types of endothelial cells could help to understand endothelial cell-related liver function.

As such, the lymphatic vascular system in the liver is a large open area for investigation¹⁶⁰, which will significantly advance our understanding of liver physiology and pathophysiology and in turn contribute to the development of new therapeutic strategies for many liver diseases.

Synopsis (Summary).

The research of the lymphatic vascular system has advanced rapidly during the last decade and has shown its dysfunction implicated in the pathogeneses of various diseases. This review article provides an overview of the lymphatic vascular system in the liver.

Abbreviation used in this paper

VEGFR

vascular endothelial growth factor receptor

VEGF

vascular endothelial growth factor

LyEC

lymphatic endothelial cell

LYVE-1

lymphatic vessel endothelial hyaluronan receptor 1

Prox1

prospero homeobox protein 1

ADH

antidiuretic hormone

NO

nitric oxide

LSEC

liver sinusoidal endothelial cell

HA

hyaluronan

HCC

hepatocellular carcinoma

HNF

hepatocyte nuclear factor

ERR

estrogen-related receptor

LRH

liver receptor homolog

ROR

retinoic acid-related orphan receptor

EHE

epithelioid hemangioendothelioma

HBxAg

hepatitis B x antigen

HBV

hepatitis B virus

BDL

bile duct ligation

HBx Ag

Hepatitis B x antigen

HBV

hepatitis virus B

MCP-1

monocyte chemoattractant protein-1

MIP-1

macrophage inflammatory protein-1

TNF

tumor necrosis factor

IL

interleukin

ICAM-1

intercellular adhesion molecule 1

VCAM-1

vascular cell adhesion molecule 1

IFN

interferon

KLH

keyhole limpet hemocyanin

CFA

complete Freund’s adjuvant

CCl₄

carbon tetrachloride

CCC

cholangiocellular carcinoma

FRC

fibroblastic reticular cell

CCL21

C-C motif chemokine ligand 21

HEV

high endothelial venules

MMR

macrophage mannose receptor 1

HSV-1

Herpes simplex virus 1

LV

lymphatic vessel

FGF-2

fibroblast growth factors-2

LPS

lipopolysaccharide

HGF

hepatocyte growth factor

LT

lymphotoxin

PI3K

phosphatidylinositol-4,5-bisphosphate 3-kinase

mTOR

mammalian target of rapamycin

TGF

tumor growth factor

STAT

signal transducer and activator of transcription