this section will address the structure of the lymphatic vascular system in general , followed by structural features specific to the liver . a detailed description of the anatomic structure of the lymphatic and hepatic lymphatic vascular systems is available in other review articles.3 , 10 , 11 , 12 lymphatic fluid originates from plasma components leaked from blood capillaries into the interstitium and then enters 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 . 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 , facilitating fluid intake in conditions of tissue swelling . lymphatic capillaries coalesce into collecting vessels , which are covered with smooth muscle cells and have basement membranes . collecting vessels lack the discontinuous junctions typical of lymphatic capillaries and are thus much less permeable . collecting vessels can be divided into smaller functional units called lymphangions that have unidirectional bicuspid valves at each end . the phasic contraction of smooth muscle cells covering lymphangions enables collecting vessels to act as pumps to drive lymphatic flow . stimulation of smooth muscle cells causes depolarization of cell membrane and opens ca channels , resulting in ca influx and smooth muscle cell contraction . smooth muscle cells also have stretch - activated ca channels that facilitate phasic contraction.19 , 20 on the other hand , lyecs produce the vasodilator nitric oxide ( no ) in response to shear stress from fluid flow , counteracting ca - dependent contraction.21 , 22 spatiotemporal alterations of ca and no levels are thereby believed to modulate the phasic contraction of lymphangions . antigen - presenting cells including dendritic cells and macrophages in lymphatic fluid interact with lymphocytes in lymph nodes , facilitating adaptive immune responses . after reaching primary lymph  nodes , lymphatic fluid flows to secondary central lymph nodes , tertiary central lymph nodes , and finally lymph trunks . lymphatic fluid from the left side of the body , abdomen , and lower limb ultimately drains into the thoracic duct , the largest lymphatic vessel , which is connected to the left subclavian vein ( figure  1 ) , whereas lymphatic fluid from other parts of the body drains into the right lymph trunk , which is connected to the right subclavian vein . lymphatic fluid that enters the subclavian veins returns to the systemic blood circulation . a schematic diagram of the hepatic lymphatic system is shown in figures  1 and 2 . unlike other tissues , the liver has sinusoids instead of capillaries . sinusoids , similar to lymphatic capillaries , are distinct from blood capillaries in that they consist of 1 layer of liver sinusoidal endothelial cells ( lsecs ) and lack basement membranes . hepatic lymphatic fluid is thought to originate from plasma components filtered through the fenestrae of lsecs into the space of disse , the interstitial space between lsecs and hepatocytes.10 , 11 fluid in the space of disse primarily flows through the space of mall , a space between the stroma of the portal tract and the outermost hepatocytes , into the interstitium of the portal tract and then into lymphatic capillaries . some portion of the 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 vein , or underneath the hepatic capsule ( figure  2 ) . lymphatic capillaries in the portal tract coalesce into collecting vessels and drain to lymph nodes at the hepatic hilum , whereas lymphatic vessels along the central vein converge into 56 large lymphatic vessels that traverse along the inferior vena cava through the diaphragm toward posterior mediastinal lymph nodes . lymphatic fluid running underneath the capsule of the convex surface of the liver drains to mediastinal lymph nodes through the coronary ligament , whereas that fluid running along the concave surface drains to lymph nodes in the hepatic hilum and to regional lymph nodes ( figure  1).10 , 11 , 12 , 29 on the basis of 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 , 11 , 12 , 29 lymphatic vessel markers generally refer to those specific to lyecs . the markers lyve-1,7 , 30 , 31 podoplanin,9 , 32 prox1,8 , 33 , 34 , 35 and vegfr-3 are most commonly used for microscopic imaging of lymphatic vessels . identification of more specific markers for the liver is needed because the most common lyec markers , lyve-1 and prox1 , are also expressed in lsecs and hepatocytes , respectively . lyve-1 is a lymphatic vessel endothelial hyaluronan ( ha ) receptor , a homolog of the cd44 ha receptor , that belongs to the superfamily of link proteins containing a conserved ha - binding domain known as the link module . its structural features suggest that 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 the fine filopodia of growing vessels during lymphangiogenesis . 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 villi , leading to death due to sepsis within 60 hours after lyve-1 depletion . 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 . compensatory mechanisms in the setting of congenital loss of lyve-1 may explain the relatively mild phenotype of these mice . in the liver , lyve-1 is expressed not only in lyecs but also in lsecs , as shown in mice and humans.40 , 41 , 42 , 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 . prox1 , a homolog of the drosophila melanogaster homeobox gene prospero , is a transcription factor and regulates genes related to lyecs , including vegfr-3 and podoplanin.45 , 46 prox1 is essential for the development of the lymphatic vascular system and also plays a role in the development of other tissues , including the lens,34 , 47 retina , heart , central nervous system , pancreas , and liver.52 , 53 prox1 is expressed in the nucleus in contrast to other lymphatic markers that are expressed in the cytoplasm or on the plasma membrane . prox1 is essential for budding of lymphatic endothelial sacs ; prox1 mice lack a lymphatic vascular system and die at approximately e14.5 . prox1 heterozygote mice die a few days after their birth and demonstrate dysfunction of lymphatic vessels with chylous ascites.8 , 33 , 47 several lines of prox1 promoter - directed reporter mice have recently been established as research tools ( gfp , morange , and tdtomato56 , 57 , 58 ) . in the early endoderm , prox1 expression is restricted to the primordia of the liver and pancreas . prox1 regulates hepatocyte migration during liver morphogenesis and is expressed in postnatal hepatocytes , although not in postnatal pancreas . in humans , cholangiocytes of normal livers were negative for prox1 expression , but intrahepatic cholangiocarcinoma and ductular cells in fibrotic septa of cirrhotic livers and hcc were positive . in addition , expression levels of prox1 ( like lyve1 ) in human hcc negatively correlated with the overall survival of patients . prox1 acts together with nuclear receptors , such as hepatocyte nuclear factor 4 , estrogen - related receptor ,62 , 63 liver receptor homolog-1 , and retinoic acid - related orphan receptors / , and regulates bile acid synthesis and circadian metabolism in the liver.63 , 65 podoplanin is a type i transmembrane glycoprotein essential for the development of the heart,66 , 67 , 68 , 69 lung , spleen , and lymph nodes . 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 . these mice have congenital lymphedema caused by  lymphatic vessel defects , although blood vessel formation is normal . podoplanin heterozygote mice are healthy and fertile , with a partial incomplete lymphatic vessel network . keratinocyte - specific podoplanin - deficient mice and a tamoxifen - inducible podoplanin depletion mouse model ( pdpn , cagcre ) have recently been developed . histologic analysis of normal mouse livers showed expression of podoplanin in cholangiocytes in addition to  lyecs . in humans , podoplanin - positive lymphatic vessels were increased in the livers of patients with decompensated cirrhosis , and podoplanin - positive fibroblastic reticular cells were increased in livers of patients with primary biliary cirrhosis . podoplanin has proven to be a useful histologic marker for diagnosing patients who have vascular tumors with lymphatic differentiation , such as epithelioid hemangioendotheliomas ( ehes ) and angiomyolipomas . vegfr-3 is a membrane - anchored tyrosine kinase and the receptor for vegf - c and vegf - d . it plays a crucial role in lymphangiogenesis . in early embryogenesis before 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 . vegfr-3 mice have lymphatic vessel defects and die at approximately e10.5 , whereas vegfr-3 heterozygous mice present with leaky lymphatic vessels and transient chylous ascites.80 , 81 a mouse line ( vegfr3 ) in which a dual reporter for fluorescence and luminescence is expressed under vegfr-3-promoter was established recently , enabling luminescence imaging of tumor - induced lymphangiogenesis . vegfr-3 is expressed by cholangiocytes in normal rat livers and is increased in cholestatic rat livers after bile duct ligation . hepatic progenitor cells were also found to express vegfr-3 in patients with primary biliary cirrhosis . hepatitis b x antigen ( hbx ag ) , one of the antigens of hepatitis b virus , 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-3positive hcc was worse than for those with vegfr-3negative hcc . 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 and then summarizes the implications of lymphangiogenesis in the pathophysiology of liver diseases . in the postnatal stage , lymphatic vessels are mostly quiescent , and lymphangiogenesis generally occurs in pathologic conditions such as tissue repair , inflammation , and tumor - related conditions . many cytokines and growth factors have been reported to promote lymphangiogenesis or inhibit lymphangiogenesis , as summarized in table  2 . the extent and duration of lymphangiogenesis are determined by the balance between pro- and anti- lymphangiogenic factors.87 , 88 signaling pathways in lymphangiogenesis 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 ) . vegf - c or vegf - d binding to vegfr-3 results in autophosphorylation of multiple c - terminal tyrosine residues in vegfr-3 , which transduces signals through the ras / raf / mek / erk pathway . signal transduction also occurs through the pi3k / akt pathway , which causes phosphorylation of akt , thereby activating mammalian target of rapamycin ( mtor ) and rac1 . chronic inflammation and malignant tumors in the liver 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 pathologic conditions remains to be demonstrated ( figure  3 ) . excellent review articles are available detailing signaling pathways in lymphangiogenesis.93 , 94 , 95 adaptive immune responses are initiated by the migration of immune cells to inflamed sites where they 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 . an increase in lymphatic vessels helps infiltrating immune cells exit inflamed sites via lymphatic vessels and accelerates resolution of inflammation.97 , 98 , 99 among the various immune cells , macrophages interact most with lymphatic vessels . lyecs secrete chemotactic factors , such as c10 , monocyte chemoattractant protein-1 , and macrophage inflammatory protein-1 , to attract macrophages . macrophages in turn secrete lymphangiogenic cytokines such as vegf - c , vegf - d , and vegf - a , which promote tumor - associated lymphangiogenesis and inflammation - induced lymphangiogenesis , as shown in the cornea , skin , and tail . macrophages were recently suggested to have the ability to transdifferentiate to lyecs.105 , 106 , 107 however , this is controversial and requires further investigation . upregulation of inflammatory cytokines such as tumor necrosis factor- and interleukin ( il ) 1 in inflamed tissues promotes expression of chemokines ( eg , ccl21/ccl19 and cxcl12 ) and their receptors ( eg , ccr7 and cxcr-4 ) in lyecs and dendritic cells,108 , 109 , 110 , 111 which enhances transmigration of dendritic cells through lyecs.112 , 113 inflammatory cytokines also increase expression of adhesion molecules such as intercellular adhesion molecule 1 , vascular cell adhesion molecule 1 , and e - selectin in lyecs and promote dendritic cell transmigration to lymphatic vessels . dendritic cells have also been reported to secrete vegf - c and promote lymphangiogenesis . in a mouse model of tail lymphedema , nude mice exhibited less edema than wild - type mice , concomitant with decreased lymphangiogenic cytokines and increased anti - lymphangiogenic cytokines . b cells promote lymphangiogenesis in inflamed lymph nodes by secreting a robust amount of vegf - a in mice given keyhole - limpet hemocyanin emulsified in  complete freund s adjuvant ( an experimental model of  inflamed lymph nodes ) . another study that used transgenic mice overexpressing vegf - a specifically in b cells showed increased lymphangiogenesis as well as angiogenesis . neutrophils are reported to contribute to lymphangiogenesis by modulating the bioavailability and bioactivity of vegf - a and by secreting vegf - d . the bioavailability of vegf - a is increased by the secretion of matrix metalloproteinases 9 and heparanase . depletion of neutrophils in mice resulted in skin inflammation in response to immunization or contact hypersensitization , and 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 . because 25%50% of lymph passing through the thoracic duct originates in the liver,1 , 120 the liver can be considered the most important organ for lymphatic fluid production . a small number of studies have reported on hepatic lymphangiogenesis in pathologic conditions such as chronic hepatitis , liver fibrosis / cirrhosis , portal hypertension , malignant tumors , and post - transplantation . resistance to sinusoidal blood flow increases in cirrhotic livers because of architectural deformations including around the portal and central venules . consequently , sinusoidal hydrostatic pressure is elevated , and plasma components filtrated through sinusoids ( which form lymphatic fluid ) increase . in cirrhotic patients , lymphatic fluid produced in the liver increases up to 30-fold,121 , 122 , 123 , 124 , 125 and liver surface lymphatic vessels dilate , as shown by peritoneoscopic observation . ascites formation in association with cirrhosis is one of the most recognized clinical manifestations of lymphatic vascular disorders . although several theories have been put forward,127 , 128 , 129 the most accepted currently is  the peripheral arterial vasodilation theory  , also known as the forward theory.130 , 131 , 132 according to this theory , splanchnic arterial vasodilation caused by portal hypertension results in underfilling of the splanchnic arterial circulation ( hypovolemia ) . in moderate stages , the hypovolemia is compensated for by renal retention of sodium and water . however , in severe portal hypertension with splanchnic arterial vasodilation , sodium and water retention is persistent and leads to leakage of fluid into the peritoneal cavity . when its  amount exceeds the absorption capacity of lymphatic vessels , ascites results.129 , 133 on a related note , impaired lymphatic drainage in the splanchnic and peripheral regions was reported in cirrhotic rats with ascites . this was accompanied by increased activity of endothelial no synthase and production of no by lyecs in these regions . in addition , smooth muscle cell coverage of lymphatic vessels in these regions was significantly decreased . treatment of these cirrhotic rats with an no synthase inhibitor improved lymphatic drainage , decreased ascites volume , and increased smooth muscle cell coverage . this study thus demonstrates a role for no in the impairment of lymphatic vessels in splanchnic and peripheral regions and in the development of ascites . it is not known whether lymphatic vessels in human cirrhotic livers show similar pathologic features . the occurrence of hepatic lymphangiogenesis was reported for the first time in liver fibrosis and cirrhosis by vollmar et  al in 1997 . they found lymphatic vessels to be increased and enlarged in rat liver cirrhosis induced by carbon tetrachloride ( ccl4 ) . these observations were confirmed the following year in patients with chronic viral hepatitis / cirrhosis . microarray analysis demonstrated a 4-fold increase in vegf - d expression in endothelial cells from ccl4-induced cirrhotic rat livers as compared with control rat livers . because vegf - d is a well - known lymphangiogenic factor that binds to vegfr-3 , which is also highly expressed in the lyecs of these cirrhotic rats , increased vegf - d could be associated with the lymphangiogenesis observed in liver cirrhosis ( figure  3 ) . it was presumed that increased lymph production that was due to increased portal pressure caused lymphangiogenesis . in 2 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 . however , the significance and mechanism of hepatic lymphangiogenesis , including in chronic hepatitis and liver fibrosis and cirrhosis , remain unknown . lymphatic vessels play a pivotal role in the pathogenesis of malignant tumors by serving as a pathway through which tumor cells metastasize . the prognosis of tumor - bearing patients with lymph node metastasis is worse than in cases without such metastasis.140 , 141 many malignant tumors 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 , and many studies have demonstrated that tumor - associated macrophages play a vital role in lymphangiogenesis in malignant tumors by secreting vegf - c and vegf - d.102 , 143 , 144 , 145 in intrahepatic cholangiocarcinoma , the lymphatic vessel density of surgically resected tumors was positively correlated with the incidence of lymphatic metastasis . in hcc , vegf - c expression was positively correlated with the size of tumors and the number of extrahepatic metastases and was negatively correlated with disease - free survival time . thus , blockade of vegf - c may be a potential therapeutic strategy against malignant tumors . a vegf - c neutralizing antibody ( vgx-100 ) is now the subject of a phase i clinical trial for adult patients with advanced or metastatic solid tumors ( nct01514123 ) . in solid organ transplants , lymphatic vessel connections between the graft and the recipient are interrupted . because lymphatic vessels are essential for adaptive immunity , post - transplant lymphangiogenesis in grafts was associated with acute cellular graft rejection in transplants of various organs ( kidney,149 , 150 , 151 heart , and lung ) in humans . however , the pathologic role of post - transplant lymphangiogenesis in graft rejection remains unclear . 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 . on the other hand , these newly formed lymphatic vessels could be beneficial if they efficiently clear immune cells . in a rat model of liver transplantation , post - transplant lymphangiogenesis in grafts was associated with long - term survival of recipients for more than 90 days . in addition , rats that had 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 have an important role 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 post - transplant period , may help increase transplant success . the lymphatic vascular system has been poorly studied in the liver . to drive research in this area , it is essential to identify better markers for lyecs that do not overlap with markers for lsecs , hepatocytes , and other liver cells . the development of experimental models for studying the lymphatic vascular system in postnatal livers will be important in examining its role and molecular mechanisms in physiological and pathophysiological conditions . although this field is wide open , it may be helpful to identify specific questions particularly in need of study . the vegf - c / vegfr-3 axis is considered the most potent signaling pathway that regulates lymphangiogenesis in other organs . however , cellular sources of vegf - c and vegfr-3 have not been fully identified in the liver . furthermore , as shown in table  2 , many other molecules are reported to regulate lymphangiogenesis . second , although the relationship between the lymphatic vascular system and metastasis is well - known and the growth of lymphatic capillaries in liver tumors has been observed , the role of lymphatic capillary growth in the development and progression of liver tumors is largely unknown . as for angiogenesis , it would be interesting to investigate lymphangiogenesis in liver cancer . third , inflammation is closely related to the development of many liver diseases , and infiltrating immune cells are drained to lymphatic vessels . thus , it would be interesting to examine lymphangiogenesis in relation to inflammation in the liver . it is also unknown how immune cells recognize lymphatic vessels at the time of migration . elucidation of these mechanisms may help in the development of anti - inflammatory strategies that facilitate immune cell clearance . fourth , although lyecs are derived from cardinal veins8 , 81 and lsecs are derived from the septum transversum , lyecs and lsecs have many similarities . both lyecs and lsecs express lyve-1.40 , 41 , 42 , 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 both lyecs and lsecs.156 , 157 furthermore , under normal conditions , neither lyecs nor lsecs are associated with basement membranes . examining the similarities and differences between these 2 types of endothelial cells could help to understand endothelial cell  related liver function . in summary , more research 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 .
the lymphatic vascular system has been minimally explored in the liver despite its essential functions including maintenance of tissue fluid homeostasis . 
 the discovery of specific markers for lymphatic endothelial cells has advanced the study of lymphatics by methods including imaging , cell isolation , and transgenic animal models and has resulted in rapid progress in lymphatic vascular research during the last decade . 
 these studies have yielded concrete evidence that lymphatic vessel dysfunction plays an important role in the pathogenesis of many diseases . 
 this article reviews the current knowledge of the structure , function , and markers of the hepatic lymphatic vascular system as well as factors  associated with hepatic lymphangiogenesis and compares liver lymphatics with those in other tissues .