Liver sinusoidal endothelial cells - The liver's gatekeeper

Zi-Bing Qian, Qi Zhang, Pei-Yun Zeng, Zhi-Xiu Zhang, Jing Liu✉

1. Gansu University of Traditional Chinese Medicine,Lanzhou 730000,China

2. Department of Endocrinology,Gansu Provincial People's Hospital,Lanzhou 730000,China

Keywords:Hepatic sinusoidal endothelial cells Immunity Chronic liver disease Inflammation Senescence

ABSTRACT Hepatic sinusoidal endothelial cell (LSECs), as the sinusoidal capillary channel of the liver,is the most abundant non-parenchymal cell group in the liver. LSEC not only forms a barrier in the hepatic sinusoid, but also has important physiological and immunological functions,including filtration, endocytosis, antigen presentation and leukocyte recruitment. It has been clear that LSECs plays an important role in maintaining immune homeostasis in the liver and delaying the immune response to acute and chronic liver injury. In this review, we summarize how LSECs affects the immune microenvironment in the liver, and discuss their role in immune-mediated chronic liver disease, carcinogenesis, inflammation and aging.

1. Summary of hepatic sinusoidal endothelial cells

LSECs is a unique and highly specialized endothelial cell in vivo. Different from the structure and function of typical vascular endothelial cells, LSECs is composed of windowed pores, which shows high endocytosis and plays an important role in maintaining the overall homeostasis of the liver. More and more studies have confirmed that LSEC dysfunction is a key event in chronic liver disease, but its role in liver disease and its significance in diagnosis,prognosis and treatment are still ignored. Under physiological conditions, LSECs is the gatekeeper of liver dynamic balance.Therefore, improving the function of LSEC seems to be a promising way to prevent the progression and complications of liver disease.Hepatocytes are mainly composed of hepatocytes, LSECs, hepatic stellate cells (HSC) and Kupffer cells (KC), which accurately "talk"to each other and influence each other. LSECs is the most permeable endothelial cell, which plays an important role in maintaining the stability of the liver environment due to the presence of window holes and the lack of basement membrane[1].Sinus cells are separated from parenchyma cells in Disse space and contain extracellular matrix (Extracellular matrix,ECM) components. Therefore, the liver microenvironment can be described as a multidirectional interaction complex (cell-matrix-cell), in which each part plays a vital role. These sinus endothelial cells are exposed to blood from the intestines and systemic circulation, meaning they are an ideal place to remove and circulate proteins and lipids in the bloodstream.Virus particles, advanced glycation end products and modified low density lipoprotein and cholesterol can be removed from the bloodstream in a matter of minutes[3].The vascular structure of the human liver is formed at 17-25 weeks of gestation, but different blood vessels in the liver have different embryonic origins. The portal vein comes from the yolk vein and the hepatic sinusoid comes from the diaphragmatic capillaries. At the 20th week of pregnancy,its unique windowing phenotype is obtained under the control of GATA binding protein 4 (GATA 4)[2].Many studies have found that vascular endothelial growth factor (VEGF) is an important factor in regulating fenestration[2].LSECs hole is a dynamic structure, and its diameter is affected by intraluminal blood pressure, hormones,drugs, toxins, changes in extracellular matrix, disease, senescence and exposure to environmental pollutants such as arsenic[3,4].Toxins and diseases are either accompanied by varying degrees of windowing, leading to gap formation, or conversely, leading to endothelial capillarization. These holes act as a "dynamic filter" that allows macromolecules to enter the parenchymal cells, and these holes may also allow viruses from the circulatory system to enter the hepatocytes[5].Animal studies show that 40% of LSECs is composed of pores, and the size and distribution of pores vary with the banded distribution of endothelial cells and the distribution of endothelial surface[6].Paracrine and autocrine cell signals are also needed to maintain LSECs windowing. VEGF from adjacent hepatocytes and stellate cells plays a key role in this regulation, and plays a role through nitric oxide (NO)-dependent and independent pathways [7].Hepatocyte group is a key regulator of hepatic microcirculation and plays a key role in hepatic sinusoid crosstalk. The characteristics of LSECs (small cells without diaphragm and basement membrane)make it an important part of the hepatocyte interaction network.The study of LSEC is very important to understand the progress,aggravation and resolution of liver disease, and the coordination of hepatocyte interaction may contribute to the development /improvement of the disease.

2. Regulation of LSEC on hepatic microcirculation

LSECs has been identified by high-resolution in vivo microscopy as swelling or contraction in response to vasoactive substances [8].The hepatic sinusoid has a dual blood supply, receiving blood flow from the portal vein (70%) and the hepatic artery (30%). Blood pressure is balanced in the sinusoid, and then blood flows into the hepatic vein and inferior vena cava [9]. In the protruding nucleus of the vascular lumen LSECs, white blood cells may temporarily block blood vessels and block blood flow, and highly malleable red blood cells usually pass through these areas easily unless the lumen shrinks or is close to zero [10]. LSECs can produce a variety of vasoactive substances, including prostaglandinI2 (PGI2) and E2 (PGE2),thromboxane A2 (TXA2), endothelin-1 (ET-1) and NO [10]. LSECs usually does not express selectin, but LSECs expresses intercellular adhesion molecule (ICAM-1). The gene encodes a cell surface glycoprotein, which is usually expressed on endothelial cells and immune system cells. ICAM-1, together with vascular cell adhesion molecule-1 ( VCAM-1) is up-regulated by inflammation, resulting in an increase in leukocyte adhesion to the endothelial surface [10].

3. LSECs immune response

The premise of liver immunity is to ensure that harmful immune responses do not precipitate on harmless antigens and that invasive pathogens can be eliminated. The first site of exposure to these antigens occurs in the hepatic sinusoid. KCs and LSECs absorb and eliminate soluble antigens entering the portal vein and play an important role in the nature of any immune response triggered by these antigens[1]. The initial key step of immune response is the inherent antigen uptake pathway of pattern recognition receptors, which are highly conserved in evolution [11].LSEC has high endocytosis with the aid of scavenger receptor (Scavenger receptors,SR), such as SR-A, SR-B (SR-B1 and CD-36), SR-E(Lox-1 and mannose receptor) and SR-H (stable protein) [12].Other high-affinity receptors that mediate endocytosis include FC γ RIIb, which contributes to the removal of antibody-mediated immune complexes. With strong lysosomal activity, LSECs plays an important role in the uptake and degradation of macromolecules and small colloids (＜ 280nm) of many blood wastes.At present, seven Toll-like receptors have been studied in LSECs, which are involved in the recognition and clearance of pathogen-related molecular patterns (PAMPs) and damage-related molecular patterns (DAMP).Like other SR, LSECs is juxtaposed with hepatocytes and plays an important role in maintaining lipid homeostasis with low density lipoprotein receptor-related protein-1 (LRP-1). LSECs co-expressed two surface lectins, namely L-specific intercellular adhesion molecule-3 capture non-integrin receptor (L-SIGN) and hepatic sinusoid endothelial cell lectin (LSECtin). LSEC also expresses several adhesion molecules, which are involved in the recruitment of leukocytes in inflammatory sites. Pattern recognition receptors include Toll-like receptor (TLR) family and scavenger receptor [11]. A typical example of how the liver regulates inflammation and immune response is the recognition of TLR4 ligand lipopolysaccharide (LPS)by KCs and LSECs. Studies on TLRs have shown that LSECs can respond to signals mediated by TLR1-TLR4, TLR6, TLR8 and TLR9, but their activation has a cell-specific response, which is limited compared with classical antigen-presenting cells, so it is helpful to resist the organ-specific response in the environment of antigen and liver drug resistance [11].

3.1 Endocytosis of LSECs

LSECs is one of the cells with the highest endocytosis ability in the human body. This property, combined with strong lysosomal activity,enables LSECs to remove waste from the blood. The endocytosis of LSECs contains different high affinity endocytosis receptors,including scavenger receptors (SR-A, SR-B and SR-H), mannose receptors and Fc γ Ⅱ b2 receptors [13].

3. 1.1 LSEC scavenger receptor

Studies in the past 10 to 15 years have confirmed that the scavenging function of LSEC mainly depends on the ability of stable protein 1 (SR-H1) and stable protein 2 (SR-H2) to mediate the binding and metabolism of multiple ligands [14]. The study found that after acetylated LDL (AcLDL) and other polyanions were ingested and metabolized by macrophages, the term "SR" was coined to denote this receptor [10]. LSEC-stabilized ligands are the products of natural renewal and remodeling in our tissues, including type I and III procollagen N-terminal propeptides, coagulation products,hyaluronic acid and chondroitin sulfate. In addition, LSEC stabilizers can effectively remove advanced glycation end products (AGEs) and oxidized low density lipoprotein (oxLDL) from circulation [14].

3.1.2 Mannose receptor and FcγⅡ b2 receptor

Mannose receptors are not specific receptors for LSECs, but bind to a wide range of glycoproteins and microbial polysaccharides, such as collagen chains (I, II, III, IV, V, XI), tissue plasminogen activators and lysosomal enzymes that regulate fibrinolytic activity, which are recruited for LSECs [15]. Therefore, they play a role in both immune and glycoprotein homeostasis. FcγⅡ b2 receptor is the only Fcreceptor expressed by LSECs. Its expression level is significantly negatively correlated with serum levels of blood lipids (triglyceride,total cholesterol, high density lipoprotein cholesterol). The loss of FcγⅡ b2 receptor in LSECs is related to liver tumorigenesis. The expression of FCγRIIb in biopsy specimens of nonalcoholic fatty liver (NAFLD) and nonalcoholic fatty liver (NASH) was studied.The results showed that there was a moderate negative correlation between serum type Ⅳ collagen and hyaluronic acid and the expression of Fc γRIIb [16]. Some studies showed that the platelet count was positively correlated with the expression of FcγRIIb on LSEC by regression analysis, suggesting that the platelet count was lower in the stage of high fibrosis, thus reducing the expression of FCγRIIb [17].

4. LSECs-pathophysiological status related to liver disease

Many pathophysiological conditions of LSECs, such as hepatic sinusoid capillarization, angiogenesis, vascular smooth muscle signal and vasoconstriction, are closely related to the occurrence and development of chronic liver diseases.

4.1 Capillarization of hepatic sinusoid

The term "capillarization" means that the unique and highly specialized phenotype of LSECs disappears, and cells become ordinary non-specialized endothelial cells, or endothelial cells of ordinary capillaries. Capillarization of LSECs occurs in both animal models and human liver injury, which is also called dedifferentiation.Capillarization occurs in the early stage of liver injury because it precedes the activation of hepatic stellate cells and macrophages and liver fibrosis, suggesting that it may be a necessary initial step in fiber formation [18]. The cause of hepatic sinusoid capillarization is not clear, but we can speculate that it is related to excessive dietary nutrition, including lipids, carbohydrates and intestinal microbial derivatives [3].In this study, it was found that the LSECs window size (frequency, porosity and diameter) was negatively correlated with the level of circulating free fatty acid (FFA) [3]. In the mice with deletion of plasma membrane vesicle-associated protein (PLVAP), the number of LSECs window was significantly decreased under transmission and scanning electron microscopy. The deletion of PLVAP leads to the obvious removal of windowing hole of LSECs, which gradually damages the chylous particle residual channel between hepatic sinusoid and hepatocytes, and then leads to hepatocyte injury [19]. Studies on the capillarization and mutual mechanism between hepatic stellate cells and hepatic stellate cells have been clearly studied in other aspects [11]. In short, LSECs can maintain the quiescent state of hepatic stellate cells as long as it is differentiated, so the differentiated LSECs is the gatekeeper of fibrosis.

4.2 Angiogenesis

Angiogenesis refers to the development of new blood vessels from pre-existing blood vessels. Hepatic angiogenesis occurs in the process of hepatic fibrosis, and these two processes are closely related. Hepatic fibrosis can promote angiogenesis, and angiogenesis can aggravate hepatic fibrosis, which has been proved by the anti-fibrotic effect of most anti-angiogenic drugs in animal models of hepatic fibrosis. However, analyzing the relationship between angiogenesis and fibrogenesis is not simple, because most of the tools used to inhibit angiogenesis also act on fibrogenesis.For example, VEGF, the main regulator of angiogenesis, is also associated with fibrogenesis [20]. Pathological angiogenesis is intrinsically linked to the progression of fibrosis in chronic liver disease, which eventually leads to liver cirrhosis and related complications, including hepatocellular carcinoma.

4.3 Vascular smooth muscle signal and vasoconstriction

LSECs can regulate fibrosis by releasing vascular smooth muscle signals. Recent studies have found that LSECs can release different vascular smooth muscle signals and balance liver regeneration and fibrosis. After acute liver injury, the activation of C-X-C chemokine receptor type 7 chemokine receptor 7 (CXCR7) pathway in LSECs stimulates the production of hepatic artery active vascular smooth muscle factor, which leads to liver regeneration. In contrast, chronic injury can lead to continuous activation of fibroblast growth factor receptor-1 (FGFR1) in LSECs, interfere with CXCR7-Id1 pathway, and tend to promote fibrogenic angiogenesis driven by CXCR4, thus leading to liver fibrosis. Therefore, different injury,differentially initiated LSECs uses different vascular smooth muscle signals to balance liver regeneration and fibrosis [21]. Endothelial cell dysfunction occurs in the early stages of chronic liver disease,even before fibrosis and inflammation, and persists in advanced liver cirrhosis [21]. Pharmacological strategies to improve LSECs in chronic liver disease, including statins, to reduce liver fibrosis,endothelial dysfunction and portal pressure [22].

5. The role of LSECs in the progression of chronic liver disease

5.1 Hepatitis C

LSECs participates in the recruitment and localization of hepatitis C effector T cells through ICAM1. The expression of VCAM1 and vascular adhesion protein 1 and CXCR3 ligand is related to the separation in parenchyma. LSECs also contributes to the retention of CXCR6+ T cells through the expression of ligand CXC- chemokine ligand 16 (CXCL16)[23]. Recent studies have found that hepatitis C core protein can down-regulate the activity of LSEC, and then reduce the synthesis and expression of adiponectin, resulting in LSEC contraction and hepatic sinusoid capillarization, increase oxidative stress, and finally lead to the activation of hepatic stellate cells [24].

5.2 Primary sclerosing cholangitis

Primary sclerosing cholangitis (PSC) and autoimmune hepatitis are liver complications associated with inflammatory bowel disease(IBD). TThe expression of mucosal address cell adhesion molecule-1(MAdCAM-1) on mucosal endothelial cells is a prerequisite for the occurrence of IBD, and it can also be detected in the hepatic vessels of patients with IBD-related liver diseases [25]. The abnormal expression of MAdCAM-1 in the liver leads to the recruitment of effector cells, which are initially activated to the liver in the intestinal tract and drive liver injury in the liver. LSECs plays a role in the abnormal homing of mucosal effector lymphocytes through the expression of MAdCAM-1 and CC- chemokine receptors 25(CCL25). VAP1 regulates the expression of MADCAM1 mainly through nuclear factor-KB (NF-KB) pathway [24].

5.3 Non-alcoholic fatty liver

NAFLD is associated with obesity, insulin resistance and / or type 2 diabetes and other metabolic abnormalities, collectively referred to as metabolic syndrome. Several independent research teams reported that in PLVAP-deficient mice, a significant decrease in the number of LSECs holes was observed, accompanied by a decrease in sinus permeability, while spontaneously developing into extensive steatosis[19]. Some studies support the view that the phenotypic change of LSECs is one of the key events in the progression of human NAFLD, suggesting that the progression of NAFLD inflammation,liver fibrosis and liver lipid metabolism disorders may affect the expression of Fc γ RIIb and macrophage function in LSECs [16].Autophagy is a physiological process that controls the dynamic balance of endothelial cells in extrahepatic vascular bed. It has been proved that there is a defect in the autophagy function of hepatic endothelial cells in patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis in the early stages of nonalcoholic steatohepatitis, but also in the late stages of chronic liver disease [25].

5.4 Hepatocellular carcinoma.

Hepatocellular carcinoma ((HCC)) is the most common chronic liver disease. In order to clarify the role of ICAM-1 in crosstalk between tumor and LSECs and hepatic stellate cell (HSC), co-culture of colorectal cancer (CRC) in vitro and liver metastasis model in vivo were studied. It was found that the interaction between ICAM-1 and LSECs and HSCs was related to tumor adhesion and angiogenesis.Blocking ICAM-1 in LSECs can reduce the adhesion and migration of tumor cells through LSEC [26]. In addition, compared with the LSECs of healthy human liver, the endothelial cells of human HCC have higher expression of integrin and lower expression of ICAM-1,and have higher ability of angiogenesis, coagulation and fibrinolysis.With the progress of HCC, the LSECs in the peri-tumor tissue will also change, such as the loss of CD32b [27]. These data suggest that peri-tumor endothelial cells play an important role in the progression of HCC, which is consistent with previous observations.

6. LSECs and inflammation

LSECs mainly regulates liver inflammation in two ways. Under inflammatory conditions, LSECs also has anti-inflammatory effects because they increase the expression of anti-inflammatory cytokine IL-10 in Th1 cells through the Notch pathway [28]. But LSECs can also be targeted by pathogens. LSECs exhibits anti-inflammatory and anti-fibrosis properties by inhibiting the activation of Kupffer cells and hepatic stellate cells and regulating intrahepatic vascular resistance and portal vein pressure. In the stage of nonalcoholic steatohepatitis, altered LSECs releases inflammatory mediators and promotes the recruitment of inflammatory cells, thereby promoting liver injury and inflammation. The altered LSECs can not maintain the stillness of hepatic stellate cells and release fibrotic mediators,thus promoting hepatic fibrosis. The increase of hepatic angiogenesis in non-alcoholic fatty liver not only promotes liver inflammation and fibrosis, but also promotes the development of hepatocellular carcinoma [29]. Recent studies have found that autophagy defects in LSECs occur in patients with NASH. In the early stage of NASH,endothelial autophagy deficiency can promote the development of liver inflammation, endothelial cell transformation to stroma,apoptosis and liver fibrosis, but it is also conducive to more advanced liver fibrosis [30]. In short, LSECs is a barrier that separates blood from the rest of the liver, thereby limiting or promoting the entry of circulating white blood cells into liver tissue.

7. Aging and LSECs

The aging process of the liver is caused by genomic and epigenome changes, which lead to the imbalance of mitochondrial function and nutrition sensing pathway, leading to cell senescence and low-grade inflammation. These changes promote multiple phenotypic changes and liver function damage in all hepatocytes (hepatocytes, sinusoidal endothelial cells, hepatic stellate cells and Kupffer cells) [31]. In particular, age-related changes in hepatic sinusoidal endothelial cells are an important but underrecognized risk factor for agerelated cardiac metabolic diseases [32]. Electron microscopic analysis showed that aging was related to the increase of the thickness of LSECs, the decrease of the number of LSEC window and the formation of basement membrane of perisinus fibrosis and central vein fibrosis. These changes reduced the porosity and endocytosis of LSECs, resulting in endothelial dysfunction. Furthermore, the clearance of chylous particulate residues is affected, resulting in lipid accumulation, postprandial hyperlipidemia and promoting the occurrence and development of atherosclerosis in the elderly[13]. Age-related liver function changes have an important impact on systemic aging and susceptibility to age-related diseases.Nutrition sensing pathway has become an important target for drug development in delaying aging and aging-related diseases.This supports the central role of liver metabolic regulation in the relationship between nutrition and aging.

8. Summary and prospect

LSECs regulates the occurrence and development of liver disease mainly through anti-inflammation, endocytosis clearance, hepatic sinusoid capillarization, secretion of angiogenic signal factors and maintenance of HSCs resting. Since the phenotypic change of LSECs is one of the earliest events of liver injury, the study of the molecular mechanism of LSECs in the context of the progression of liver injury is of great value for early detection and early intervention of chronic liver diseases. Making full use of the special immune characteristics of LSECs to formulate future immunotherapy strategies may find a new opportunity for more effective treatment of chronic liver disease.