Liver Sinusoidal Endothelial Cell Line Research Articles

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions invitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-β), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-β1 stimulation. Therefore, this hierarchical invitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Engineered silica nanoparticles are biologically safe vehicles to deliver drugs or genes to liver cells

Engineered silica nanoparticles (SiNP) are emerging materials for medical applications. Evaluating biological responses of specific cells treated with engineered silica nanoparticles is however essential. We synthesized and characterized the physicochemical properties of silica nanoparticles with two different sizes of 10 and 100 nm (10SiNP and 100SiNP) dispersed in cell culture medium. HuH-7, an epithelial-like human hepatoblastoma cell line and SK-HEP-1, a liver sinusoidal endothelial cell line (LSEC) are employed to evaluate their biological responses for the SiNP treatment. Primary human lymphocytes are used to assess genotoxicity recommended by OECD guidelines while erythrocytes are used to assess hemolytic activity. The engineered silica nanoparticles are not able to produce radical species, to alter the mitochondrial membrane potential, and induce any adverse effects on cell proliferation. The colony formation ability of HuH-7 hepatoblastoma cells was not affected following the SiNP treatment. Furthermore, SiNPs do not induce hemolysis of red blood cells and are not genotoxic. These findings suggest that SiNPs regardless of the size, amount, and incubation time are biologically safe vehicles to deliver drugs or genes to the liver.

A defect in endothelial autophagy occurs in patients with non-alcoholic steatohepatitis and promotes inflammation and fibrosis

Previous studies demonstrated that autophagy is protective in hepatocytes and macrophages, but detrimental in hepatic stellate cells in chronic liver diseases. The role of autophagy in liver sinusoidal endothelial cells (LSECs) in non-alcoholic steatohepatitis (NASH) is unknown. Our aim was to analyze the potential implication of autophagy in LSECs in NASH and liver fibrosis. We analyzed autophagy in LSECs from patients using transmission electron microscopy. We determined the consequences of a deficiency in autophagy: (a) on LSEC phenotype, using primary LSECs and an LSEC line; (b) on early stages of NASH and on advanced stages of liver fibrosis, using transgenic mice deficient in autophagy specifically in endothelial cells and fed a high-fat diet or chronically treated with carbon tetrachloride, respectively. Patients with NASH had half as many LSECs containing autophagic vacuoles as patients without liver histological abnormalities, or with simple steatosis. LSECs from mice deficient in endothelial autophagy displayed an upregulation of genes implicated in inflammatory pathways. In the LSEC line, deficiency in autophagy enhanced inflammation (Ccl2, Ccl5, Il6 and VCAM-1 expression), features of endothelial-to-mesenchymal transition (α-Sma, Tgfb1, Col1a2 expression) and apoptosis (cleaved caspase-3). In mice fed a high-fat diet, deficiency in endothelial autophagy induced liver expression of inflammatory markers (Ccl2, Ccl5, Cd68, Vcam-1), liver cell apoptosis (cleaved caspase-3) and perisinusoidal fibrosis. Mice deficient in endothelial autophagy treated with carbon tetrachloride also developed more perisinusoidal fibrosis. A defect in autophagy in LSECs occurs in patients with NASH. Deficiency in endothelial autophagy promotes the development of liver inflammation, features of endothelial-to-mesenchymal transition, apoptosis and liver fibrosis in the early stages of NASH, but also favors more advanced stages of liver fibrosis. Autophagy is a physiological process controlling endothelial homeostasis in vascular beds outside the liver. This study demonstrates that autophagy is defective in the liver endothelial cells of patients with non-alcoholic steatohepatitis. This defect promotes liver inflammation and fibrosis at early stages of non-alcoholic steatohepatitis, but also at advanced stages of chronic liver disease.

A new immortalized human liver sinusoidal endothelial cell line: Establishment, characterization, and application potential to development of in vitro human liver models

A new immortalized human liver sinusoidal endothelial cell line: Establishment, characterization, and application potential to development of in vitro human liver models

Correction: Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease.

[This corrects the article DOI: 10.1371/journal.pone.0159217.].

Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles in liver injury and repair, but their role in the underlying pathogenetic mechanisms of non-alcoholic fatty liver disease remains undefined. Here, we evaluated the effects of steatosis on LSEC gene expression in a murine model of non-alcoholic fatty liver disease and an immortalized LSEC line. Using microarray we identified distinct gene expression profiles following exposure to free fatty acids. Gene pathway analysis showed a number of differentially expressed genes including those involved in lipid metabolism and signaling and inflammation. Interestingly, in contrast to hepatocytes, fatty acids led to decreased expression of pro-inflammatory chemokines including CCL2 (MCP-1), CXCL10 and CXCL16 in both primary and LSEC cell lines. Chemokine downregulation translated into a significant inhibition of monocyte migration and LSECs isolated from steatotic livers demonstrated a similar shift towards an anti-inflammatory phenotype. Overall, these pathways may represent a compensatory mechanism to reverse the liver damage associated with non-alcoholic fatty liver disease.

Spontaneous immortalization of mouse liver sinusoidal endothelial cells.

The spontaneous immortalization of cells in vitro is a rare event requiring genomic instability, such as alterations in chromosomes and mutations in genes. In the present study, we report a spontaneously immortalized liver sinusoidal endothelial cell (LSEC) line generated from mouse liver. These immortalized LSECs showed typical LSEC characteristics with the structure of transcellular fenestrations, the expression of von Willebrand factor (VWF) and the ability to uptake DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). However, these immortalized LSECs lost the ability to form capillary-like structures, and showed clonal and multilayer growth without contact inhibition. Moreover, their proliferation rate increased with the increase in the number of passages. In addition, these cells obained the expression of CD31 and desmin, and showed an upregulation of p53 protein expression; however, their karyotype was normal, and they could not form colonies in soft agar or tumors in SCID mice. In conclusion, in the present study, we successfully established a spontaneously immortalized LSEC line.

A new reporter mouse cytomegalovirus reveals maintained immediate-early gene expression but poor virus replication in cycling liver sinusoidal endothelial cells

BackgroundThe MCMV major immediate early promoter/enhancer (MIEP) is a bidirectional promoter that drives the expression of the three immediate early viral genes, namely ie1, ie2 and ie3. The regulation of their expression is intensively studied, but still incompletely understood.MethodsWe constructed a reporter MCMV, (MCMV-MIEPr) expressing YFP and tdTomato under the control of the MIEP as proxies of ie1 and ie2, respectively. Moreover, we generated a liver sinusoidal endothelial cell line (LSEC-uniLT) where cycling is dependent on doxycycline. We used these novel tools to study the kinetics of MIEP-driven gene expression in the context of infection and at the single cell level by flow cytometry and by live imaging of proliferating and G0-arrested cells.ResultsMCMV replicated to higher titers in G0-arrested LSEC, and cycling cells showed less cytopathic effect or YFP and tdTomato expression at 5 days post infection. In the first 24 h post infection, however, there was no difference in MIEP activity in cycling or G0-arrested cells, although we could observe different profiles of MIEP gene expression in different cell types, like LSECs, fibroblasts or macrophages. We monitored infected LSEC-uniLT in G0 by time lapse microscopy over five days and noticed that most cells survived infection for at least 96 h, arguing that quick lysis of infected cells could not account for the spread of the virus. Interestingly, we noticed a strong correlation between the ratio of median YFP and tdTomato expression and length of survival of infected cells.ConclusionBy means of our newly developed genetic tools, we showed that the expression pattern of MCMV IE1 and IE2 genes differs between macrophages, endothelial cells and fibroblasts. Substantial and cell-cycle independent differences in the ie1 and ie2 transcription could also be observed within individual cells of the same population, and marked ie2 gene expression was associated with longer survival of the infected cells.

Tumorigenicity of cancer stem-like cells derived from hepatocarcinoma is regulated by microRNA-145

microRNAs are implicated in cancer initiation and progression by their ability to affect the expression of genes and proteins that regulate cell proliferation and death. Recent studies found that the stem cell-related genes Sox2, Oct4 and Klf4 are among the target genes regulated by microRNA-145 (miR-145), suggesting that miR-145 possibly plays a role in the maintenance of cancer stem cells. Therefore, it is important to address the involvement of miR-145 in the key roles of cancer stem cells in cancer initiation, progression and reoccurrence. We compared miR-145 expression in the cancer stem-like cells (T3A-A3) derived from hepatocarcinoma, in the hepatocarcinoma cell line BEL-7402 and in the normal liver sinusoidal endothelial cell line (LSEC). As demonstrated by a TaqMan microRNA real-time assay, T3A-A3 cells express lower miR-145 levels compared to the other cell lines. To address the role of miR-145 in cancer stem cells, miR-145 was restored in T3A-A3 cells. This resulted in senescence-like G1 arrest in cell cycling, and significantly inhibited clonogenic cell expansion in vitro and xenograft tumor growth in vivo. Moreover, miR-145 restoration diminished tumorsphere growth of T3A-A3 cells in vitro and T3A-A3 cells tumor formation in nude mice in vivo. Additionally, the increase in miR-145 levels paralleled the decrease in Oct4 levels. The effect of miR-145 on tumor suppression in T3A-A3 cells was partly reversed by overexpression of Oct4 both in vitro and in vivo. Collectively, our data indicate that miR-145 plays an important role in cancer stem cell tumorigenicity, potentially via modulation of the downstream target, Oct4.

Dengue virus infection of SK Hep1 cells: inhibition ofin vitroangiogenesis and altered cytomorphology by expressed viral envelope glycoprotein

Dengue virus (DENV) infection of human endothelial cells has been implicated in the pathobiology of dengue hemorrhagic fever and dengue shock syndrome. However, the mechanisms by which DENV infections alter the functional physiology of endothelial cells remain incompletely understood. In the present study, we examined the susceptibility of a human liver sinusoidal endothelial cell line SK Hep1 to all four serotypes of DENV and studied the effect of the virus on in vitro angiogenesis. All four serotypes of DENV could infect the SK Hep1 cells, but showed variable cytopathic effects, the most pronounced being that of DENV-2. Electron microscopy of the infected cells showed significant ultrastructural changes. In vitro angiogenesis assays on DENV-2 exposed SK Hep1 cells in the matrigel system showed inhibition compared with the controls. Importantly, transfection and transient expression of the DENV-2 envelope glycoprotein (E) in these cells showed drastic alterations in cell shapes and the E protein could be localized by fluorescence microscopy in terminal knob-like structures. Therefore, SK Hep1, a human hepatic sinusoid-derived endothelial cell line, may constitute a potential model to study DENV-endothelial cell interactions in vitro, especially towards understanding the possible virus-induced changes in hepatic endothelium and its role in disease pathogenesis.

Human liver sinusoidal endothelial cells respond to interaction with Entamoeba histolytica by changes in morphology, integrin signalling and cell death

Invasive infection with Entamoeba histolytica causes intestinal and hepatic amoebiasis. In liver, parasites cross the endothelial barrier before abscess formation in the parenchyma. We focussed on amoebae interactions with human hepatic endothelial cells, the latter potentially playing a dual role in the infection process: as a barrier and as modulators of host defence responses. We characterized early responses of a human liver sinusoidal endothelial cell line to virulent and virulence-attenuated E. histolytica. Within the first minutes human cells start to retract, enter into apoptosis and die. In the presence of virulent amoebae, expression of genes related to cell cycle, cell death and integrin-mediated adhesion signalling was modulated, and actin fibre, focal adhesion kinase and paxillin localizations changed. Effects of inhibitors and amoeba strains not expressing pathogenic factors amoebapore A and cysteine protease A5 indicated that cell death and cytoskeleton disorganization depend upon parasite adhesion and amoebic cysteine proteinase activities. The data establish a relation between cytotoxic effects of E. histolytica and altered human target cell adhesion and suggest that interference with adhesion signalling triggers endothelial cell retraction and death. Understanding the roles of integrin signalling in endothelial cells will provide clues to unravel host-pathogen interactions during amoebic liver infection.

Morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line

To investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line (LSEC line). Immunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line, and flow cytometry was used to analyze the purity of the human LSEC line. The morphology (including W-P bodies and surface fenestrations) and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope. The proliferation curve of the human LSEC line was analyzed by MTT assay. The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1, activities of fibrinolysis (PAI-1, t-PA, u-PA), releasing of IL-6 and IL-8 were compared respectively by enzyme linked immunosorbent assay. Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL. The established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors. Immunofluorescence staining showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF), and could take up acetylated low-density lipoproteins (Ac-LDL). The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis. The W-P bodies and the phagocytosis of Dynabeads was demonstrated by transmission electron microscope. And fenestrations could be found cellular surface with scanning electron microscopy. When compared with human primary LSEC, the human LSEC line has an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1. The human LSEC line can also release PAI-1, t-PA, u-PA but can not release IL-6 and IL-8 to TNF-alpha. In contrast, human primary LSEC could release IL-6. The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis, and the percentage of apoptotic cells was as high as (38.4 +/- 6.7)%, while (28.6 +/- 4.5)% and (7.8 +/- 1.2)% respectively in primary LSEC and in human umbilical vein endothelial cells. The established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apoptosis. Therefore it is feasible to use this cell line for the study of liver ischemia-reperfusion injury.