Modulation of lymphatic vessels in management of liver disease and complications
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Liver is the largest producer of lymph entering into the thoracic duct (TD). The hepatic lymph mainly originates from the sinusoids and transverses through intrahepatic lymphatic vessels (LVs). It drains into the local lymph nodes through LVs located in three regions of the liver, viz., portal, sublobular and capsular, with about 80% of it draining via the portal LVs [1]. In recent years, several markers have been established for the identification of hepatic LVs and lymphatic endothelial cells (LyECs), such as prospero homeobox-1 transcription factor (PROX-1), vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), surface glycoprotein podoplanin (PDPN) and interleukin-7 (IL-7) [2,3]. A right amalgamation of these markers clearly distinguishes LyECs from other liver cells like liver sinusoidal endothelial cells, Kupffer cells and cholangiocytes. During the progression of liver disease, hepatic lymph flow, LV structure, numbers and molecular phenotype of LyECs are all known to be profoundly altered. An increase in lymph flow in liver disease is due to an increased sinusoidal hydrostatic pressure and a decreased oncotic pressure, i.e., hypoalbuminemia [4]. Increased lymph flow in liver triggers sprouting of new LVs by a process termed ‘lymphangiogenesis’ that is driven mainly by growth factors, VEGF-C and VEGF-D. These growth factors bind to VEGFR-3 on LyECs and facilitate lymphangiogenesis via downstream activation of phosphoinositide 3 kinase and/or mitogen activated protein kinase pathways [5]. Irrespective of the etiology, there is an increased number of dilated hepatic PDPN+ LVs in and around the portal areas in patients with liver disease and portal hypertension [6,7]. Using a combination of markers, that includes CD45-CD31+LYVE-1+/PDPN+, hepatic LyECs have been isolated and studied by deep transcriptome sequencing in CCl4 and bile duct ligation (BDL)-induced liver fibrosis and non-fibrotic nonalcoholic steatohepatitis (NASH) mice models [2,8]. As compared to healthy mice, a decreased expression of Prox1 and tight junction protein, Zonula occludens-1 in the LyECs of NASH, CCl4 and BDL mice has been illustrated. Importantly, many extracellular matrix genes are seen to be upregulated in LyECs of CCl4 and BDL mice along with collagen deposition in portal areas, while many of these genes are decreased in NASH mice as compared to healthy mice livers, indicating a key role of dysfunctional hepatic LyECs during progression of liver fibrosis [2]. Studies by Tamburini et al. have unraveled that one of the major pathways to be upregulated in LyECs from patients with advanced NASH is the pro-fibrogenic IL-13 signalling pathway [6]. In another study on murine models of NASH, it has been shown that an increase in the density of hepatic LVs is associated with a decrease in the expression of PROX1 and VEGFR3 in LyECs. The study also reports an increase in expression of permeability regulator, VE-cadherin (Cdh5) in the hepatic LyECs along with a decrease in LV drainage to nearby lymph nodes as compared to controls. This indicates that although LyECs undergo dynamic changes in a NASH liver to induce expansion of LVs, their identity, permeability and drainage functions are compromised [8].
Besides liver, there are also alterations in the extrahepatic LVs during advanced liver disease. Ribera et al. have reported a reduction in coverage of smooth muscle cells on the collecting mesenteric LVs with impaired mesenteric lymph flow in the splanchnic regions in rat models of cirrhosis. Molecularly, this has been attributed to an overexpression of endothelial nitric oxide synthase leading to a high production of nitric oxide by cirrhotic LyECs of the mesenteric LVs [9]. In our studies, we have also reported enhanced numbers of dilated and leaky mesenteric LVs in rat models of cirrhosis along with many molecular changes in mesenteric LyECs, mainly an increased expression of Cdh5, in comparison to that observed in healthy controls [10]. Additionally, we and others have also shown a significant upregulation of dilated duodenal PDPN+ LVs and presence of lymphangiectasia in cirrhotic patients with ascites as compared to those without ascites, indicating considerable changes in the intestinal LVs and LyECs in advanced cirrhosis [11,12].
LVs from both liver and gut collect the extravasated fluid as lymph and transport it to the cisterna chyli and TD to return it to the bloodstream via the lymphovenous junction. By cannulating the TD in patients with advanced alcoholic cirrhosis, Dumont et al. [13] showed that TD lymph flow was 3 to 6-fold higher in the patients as compared to the normal rate of 1ml per minute flow in healthy subjects. On a 2-year follow-up of 76 patients with cirrhosis, a recent study has now recorded that the diameter of cisterna chyli and TD initially enlarge due to increased volume of lymph but then narrow down at a later point, particularly in patients with advanced cirrhosis [14]. The study depicted that patients with a narrow TD have higher risk of developing complications such as refractory ascites and hepatic encephalopathy. Although mechanisms and sequelae of TD narrowing remain obscure, it is logical to speculate that when hepatic and intestinal lymph flow is overwhelmed and TD is not able to return excess fluid back to the systemic circulation, the renin-aldosterone system in the kidneys gets activated, leading to abnormal renal sodium and water retention, further aggravating lymph imbalance, clinical ascites and systemic inflammation. This might be leading to extrahepatic complications of cirrhosis like hepatic encephalopathy [15]. In fact, waste clearance by brain lymphatic system, comprising of the meningeal and glial LVs, has also been shown to be functionally impaired in rat models of hepatic encephalopathy [16,17]. Mechanistically, this has been associated with a reduced expression of aquaporin 4 (AQP4), an important water transport protein in astrocytes present in the olfactory bulb and prefrontal cortex regions of the brain [17].
Improving lymphatic dysfunction in the liver and other organs is emerging as an innovative and prudent approach to manage advanced liver disease (Figure). In preclinical models of cirrhosis, the use of an eNOS inhibitor, LN(G)-methyl-L-arginine, has been shown to ameliorate lymphatic contractility and drainage in the peritoneal cavity and reduce abdominal ascites by increasing coverage of smooth muscle cells on L-yECs [9]. TD stenting in patients with refractory ascites improves lymph outflow from TD to venous circulation via lymphovenous junction and resolves ascites [18,19]. A study on cirrhotic patients with dilated cisterna chyli has reported that the diameter of the chyli is significantly decreased due to a reduction in hepatic lymph production after transjugular intrahepatic portosystemic shunt (TIPS). A decline in portal pressure after TIPS may thus be partly attributed to a reduction in hepatic lymph production and a decompression of the central lymphatic system [20]. Pro-lymphangiogenic growth factor therapy using a recombinant form of VEGF-C (rVEGF-C), in which Cys156 is replaced by a Ser residue, has proven to be one of the most promising and specific approaches to induce lymphangiogenesis. In NASH mice models, injection of rVEGF-C has been shown to induce increased proliferation of hepatic LyECs, ameliorate defective lymphatic drainage and significantly reduce liver inflammation [8]. In our studies, we have used rVEGF-C, encapsulated within lipid nanoparticles and when provided orally in cirrhotic animal models, it resulted in increased mesenteric lymphatic drainage, reduced portal pressure, ascites volume, and systemic endotoxemia [10]. Local injection of adeno-associated viral VEGF-C (AAV8-VEGF-C) in cisterna magna has also been shown to result in increased meningeal lymphatic drainage, ameliorating neuroinflammation in rats with hepatic encephalopathy [17].
To summarize, maneuvers that effectively restore lymph dynamics by either reducing lymph inflow or accelerating lymph outflow seem to have promising benefits in end-stage liver cirrhosis patients with limited treatment options. Development of novel targeted LV-modulatory therapies in the light of emerging transcriptome data specific to hepatic and extrahepatic LyECs provides us with new vistas to treat and manage liver disease and its many fatal complications.
Notes
Authors’ contribution
A.S. Figure generation, writing. P.J. Inputs in Figure and text. S.K.S. Text revision. SK. concept of the figure and manuscript draft, editing and approval of the manuscript.
Acknowledgements
The figure was created using Biorender (Created in Bio-Render. DDGG, A. (2024) BioRender.com/q26e162).
Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
BDL
bile duct ligation
IL-7
interleukin-7
LVs
lymphatic vessels
LyECs
lymphatic endothelial cells
LYVE-1
lymphatic vessel endothelial hyaluronan receptor-1
NASH
nonalcoholic steatohepatitis
PDPN
podoplanin
PROX-1
prospero homeobox-1 transcription factor
TD
thoracic duct
TIPS
transjugular intrahepatic portosystemic shunt
VEGFR-3
vascular endothelial growth factor receptor-3