Human Liver Endothelial Cells Research Articles

A genetic association study of circulating coagulation factor VIII and von Willebrand factor levels

AbstractCoagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10−9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

Organ-on-a-chip for studying immune cell adhesion to liver sinusoidal endothelial cells: the potential for testing immunotherapies and cell therapy trafficking.

Immunotherapy has changed the landscape of treatment options for patients with hepatocellular cancer. Checkpoint inhibitors are now standard of care for patients with advanced tumours, yet the majority remain resistant to this therapy and urgent approaches are needed to boost the efficacy of these agents. Targeting the liver endothelial cells, as the orchestrators of immune cell recruitment, within the tumour microenvironment of this highly vascular cancer could potentially boost immune cell infiltration. We demonstrate the successful culture of primary human liver endothelial cells in organ-on-a-chip technology followed by perfusion of peripheral blood mononuclear cells. We confirm, with confocal and multiphoton imaging, the capture and adhesion of immune cells in response to pro-inflammatory cytokines in this model. This multicellular platform sets the foundation for testing the efficacy of new therapies in promoting leukocyte infiltration across liver endothelium as well as a model for testing cell therapy, such as chimeric antigen receptor (CAR)-T cell, capture and migration across human liver endothelium.

Abstract 13203: CD36 Regulates Factor VIII Secretion From Liver Endothelial Cells

Introduction: Abnormal levels of the coagulation factor VIII (FVIII) are a risk factor for venous thromboembolism (VTE). Genome-wide association studies have identified novel candidate gene associations that may regulate FVIII levels in humans, including CD36. Hypothesis: We hypothesized that CD36 regulates FVIII release from endothelial cells. Methods and Results: We identified a subset of human liver endothelial cells (HLEC) expressing FVIII and purified them using a differentially expressed cell surface protein CD32. CD32+ HLEC expressed FVIII, transported FVIII in intracellular vesicles, and secreted FVIII. We stimulated CD36 signaling or silenced CD36 to test the effect of CD36 on FVIII release. Oxidized LDL (oxLDL), a CD36 agonist, increased endothelial release of FVIII. Conversely, silencing CD36 decreased FVIII release. We searched for an intracellular signaling pathway that mediates CD36 stimulation of FVIII secretion and found that oxLDL increased p38 activity and a p38 inhibitor decreased FVIII release. Conclusions: Taken together, our data show that CD36 stimulates FVIII release through a p38 dependent pathway in specialized liver endothelial cells. These results validate genetic epidemiology data linking CD36 to FVIII levels in humans and provide new insights into the regulation of FVIII levels.

Decellularized Liver Nanofibers Enhance and Stabilize the Long-Term Functions of Primary Human Hepatocytes In Vitro.

Owing to significant differences across species in liver functions, in vitro human liver models are used for screening the metabolism and toxicity of compounds, modeling diseases, and cell-based therapies. However, the extracellular matrix (ECM) scaffold used for such models often does not mimic either the complex composition or the nanofibrous topography of native liver ECM. Thus, here novel methods are developed to electrospin decellularized porcine liver ECM (PLECM) and collagen I into nano- and microfibers (≈200-1000nm) without synthetic polymer blends. Primary human hepatocytes (PHHs) on nanofibers in monoculture or in coculture with nonparenchymal cells (3T3-J2 embryonic fibroblasts or primary human liver endothelial cells) display higher albumin secretion, urea synthesis, and cytochrome-P450 1A2, 2A6, 2C9, and 3A4 enzyme activities than on conventionally adsorbed ECM controls. PHH functions are highest on the collagen/PLECM blended nanofibers (up to 34-fold higher CYP3A4 activity relative to adsorbed ECM) for nearly 7 weeks in the presence of the fibroblasts. In conclusion, it is shown for the first time that ECM composition and topography synergize to enhance and stabilize PHH functions for several weeks in vitro. The nanofiber platform can prove useful for the above applications and to elucidate cell-ECM interactions in the human liver.

Analysis of Neutral Lipid Accumulation and Lipotoxicity in Non-hepatocyte Liver Cells Under Free Fatty-acid Treatment

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) and its advanced form, nonalcoholic steatohepatitis (NASH), are prevalent liver diseases with no effective treatment available. NAFLD is marked by increased lipid accumulation in the hepatocytes, lobular inflammation and fibrosis. However, the effects of lipid accumulation in other hepatic lineage cells, such as liver endothelial cells (LEC), intrahepatic cholangiocytes (IHCHOLs), and hepatic stellate cells (HSCs), on NAFLD/NASH progression are less understood.&#x0D; METHODS: Human HSCs, IHCHOLs, and LECs were isolated from liver explants of healthy donors and further characterized with immunofluorescence staining. The cells were treated with different concentrations of free fatty acids (FFAs, oleic acid and palmitic acid 2:1) for 48 hours, and then examined for lipid accumulation with staining of BODIPY493/503. A cell viability assay and LDH cytotoxicity assay were performed. Real-time PCR was performed to monitor gene expression in LECs after treatment.&#x0D; RESULTS: Lipid accumulation confirmed by BODIPY493/503 staining was observed following treatment, especially with higher FFA concentrations. No statistically significant difference in cell viability or cytotoxicity of the cells was observed under different treatment concentrations of FFA. However, a negative trend in cell viability was observed with an increment of FFA dosage. FFA treatment upregulated the expression of the genes in LEC that are related to angiogenesis (CDH5), epithelial-mesenchymal transformation (TGF-β2 and TGF-β3), cellular stress (P53), inflammation (IL-32) and immunoglobular cell adhesion molecules (ICAM-1 and VCAM-1) that are key to monocyte adhesion to endothelial cells in response to a local inflammatory signal.&#x0D; CONCLUSION: Using an in vitro cellular model system, we examined lipid accumulation and cellular stress in different human liver cell types, especially LECs, under dose-dependent FFA treatment. This could be further developed to delineate adaptive responses within different hepatic lineage cells in the progression of NAFLD/NASH.&#x0D; IMPACT: A better understanding of the involvement of hepatocytes and other hepatic lineages in the progression of NAFLD/NASH will augment the development of targeted and effective treatments.

Cell surface biotinylation to identify the receptors involved in nanoparticle uptake into endothelial cells

Targeted drug delivery requires –among others- specific interaction of nanocarriers with cell surface receptors enabling efficient internalization into the targeted cells. Thus, identification of receptors allowing efficient nanocarrier uptake is essential to improve the design of targeted nanomedicines. Here we used methods based on cell surface biotinylation to identify cell surface receptors mediating nanoparticle uptake by cells. We used human brain and liver endothelial cells as representative examples of cells typically showing very low and very high nanoparticle uptake, respectively. Amino-modified and carboxylated silica were used as model nanoparticles usually associated with high and low uptake into cells, respectively, and carrying different coronas after exposure in full human plasma. Using cell surface biotinylation of live cells and receptor pull-down assays, we compared the receptors internalized in control untreated cells and those internalized upon exposure to nanoparticles. In this way, we identified receptors associated with (high) nanoparticle uptake. The candidate receptors were further validated by decorating the nanoparticles with an artificial corona consisting of the respective receptor ligands. We found that a vitronectin corona can be used to target integrin receptors and strongly enhances nanoparticle uptake in brain and liver endothelial cells. The increased uptake was maintained in the presence of serum, suggesting that the vitronectin-corona could resist interaction and competition with serum. Furthermore, plasminogen-coated nanoparticles promoted uptake in endothelial cells of the liver, but not of the brain. The presented approach using reversible biotinylation of cell surface receptors in live cells allows for receptor-based targeting of nanocarriers that are instrumental in nanoparticle uptake, which can be exploited for targeted drug delivery. Statement of significanceIn order to deliver drugs to their site of action, drug-loaded nanocarriers can be targeted to cell receptors enabling efficient uptake into target cells. Thus, methods to identify nanocarrier receptors are invaluable. Here we used reversible biotinylation of live cells and receptor pull-down approaches for receptor identification. By comparative analysis of the individual receptors internalized in untreated cells and cells exposed to nanoparticles, we identified receptors enabling high nanoparticle uptake into liver and brain endothelial cells. Their role was confirmed by decorating nanoparticles with an artificial corona composed of the receptor ligands. In conclusion, live cell reversible biotinylation of cell surface proteins is a powerful tool for the identification of potential receptors for receptor-based targeting of nanocarriers.

Abstract 14391: The Transcriptional Landscape of Liver Sinusoidal Endothelial Cells

Introduction: Human liver endothelial cells (HLEC) are a heterogeneous cell population. Single cell transcriptomics and immunostaining assays have categorized HLEC into two major groups according to their location within the liver lobules (zone 1 or periportal HLEC and zones 2/3 or central venous (CV) HLEC). Hypothesis: Cellular deconvolution of the bulk transcriptome of HLEC will highlight important properties of HLECs and their transcriptional regulators. Methods: We uniformly processed all bulk RNA-seq data from HLECs in Gene Expression Omnibus. We used CIBERSORTx with human liver single cell RNA-seq data to perform cell type deconvolution of bulk HLEC RNA-seq. We compared the transcriptome of HLEC subpopulations to that of HUVEC. We then applied the HOMER motif finding algorithm on the promoters of genes up/downregulated in HLEC compared to HUVEC. Results: We analyzed data from 10 HLEC samples and 107 HUVEC samples from 37 independent experiments. Principal component analysis revealed that 6 of the 10 HLEC samples have robust transcriptional differences compared to HUVEC. CIBERSORTx showed that those 6 HLEC samples correspond to CV HLECs and can be identified by FCGR2B expression. Differential gene expression analysis revealed enrichment of inflammatory and coagulation pathways in CV HLEC and enrichment of proliferation and mitosis pathways in HUVEC. CV HLEC showed increased expression of factors VIII, X, XII, Protein S and antithrombin III. We validated gene expression at the protein level by Western blot. A transcription factor (TF) prioritization approach using HOMER motif enrichment revealed a potential role for E2F, and RXR-RAR TF as primary determinants of the differences between HUVEC and CV HLEC. Conclusions: We identify and characterize CV HLEC as a distinct endothelial subtype relevant to inflammation and coagulation. We also identify downregulation of E2F and upregulation of RXR-RAR TF as potential key regulators of the CV HLEC transcriptome.

Development of Biomimetic Hepatic Lobule-Like Constructs on Silk-Collagen Composite Scaffolds for Liver Tissue Engineering.

Constructing an engineered hepatic lobule-mimetic model is challenging owing to complicated lobular architecture and crucial hepatic functionality. Our previous study has demonstrated the feasibility of using silk fibroin (SF) scaffolds as functional templates for engineering hepatic lobule-like constructs. But the unsatisfactory chemical and physical performances of the SF-only scaffold and the inherent defect in the functional activity of the carcinoma-derived seeding cells remain to be addressed to satisfy the downstream application demand. In this study, SF-collagen I (SFC) composite scaffolds with improved physical and chemical properties were fabricated, and their utilization for bioengineering a more hepatic lobule-like construct was explored using the immortalized human hepatocyte-derived liver progenitor-like cells (iHepLPCs) and endothelial cells incorporated in the dynamic culture system. The SFC scaffolds prepared through the directional lyophilization process showed radially aligned porous structures with increased swelling ratio and porosity, ameliorative mechanical stiffness that resembled the normal liver matrix more closely, and improved biocompatibility. The iHepLPCs displayed a hepatic plate-like distribution and differentiated into matured hepatocytes with improved hepatic function in vitro and in vivo. Moreover, hepatocyte–endothelial cell interphase arrangement was generated in the co-culture compartment with improved polarity, bile capillary formation, and enhanced liver functions compared with the monocultures. Thus, a more biomimetic hepatic lobule-like model was established and could provide a valuable and robust platform for various applications, including bioartificial liver and drug screening.

Endothelial and hematopoietic hPSCs differentiation via a hematoendothelial progenitor

BackgroundhPSC-derived endothelial and hematopoietic cells (ECs and HCs) are an interesting source of cells for tissue engineering. Despite their close spatial and temporal embryonic development, current hPSC differentiation protocols are specialized in only one of these lineages. In this study, we generated a hematoendothelial population that could be further differentiated in vitro to both lineages.MethodsTwo hESCs and one hiPSC lines were differentiated into a hematoendothelial population, hPSC-ECs and blast colonies (hPSC-BCs) via CD144+-embryoid bodies (hPSC-EBs). hPSC-ECs were characterized by endothelial colony-forming assay, LDL uptake assay, endothelial activation by TNF-α, nitric oxide detection and Matrigel-based tube formation. Hematopoietic colony-forming cell assay was performed from hPSC-BCs. Interestingly, we identified a hPSC-BC population characterized by the expression of both CD144 and CD45. hPSC-ECs and hPSC-BCs were analyzed by flow cytometry and RT-qPCR; in vivo experiments have been realized by ischemic tissue injury model on a mouse dorsal skinfold chamber and hematopoietic reconstitution in irradiated immunosuppressed mouse from hPSC-ECs and hPSC-EB-CD144+, respectively. Transcriptomic analyses were performed to confirm the endothelial and hematopoietic identity of hESC-derived cell populations by comparing them against undifferentiated hESC, among each other’s (e.g. hPSC-ECs vs. hPSC-EB-CD144+) and against human embryonic liver (EL) endothelial, hematoendothelial and hematopoietic cell subpopulations.ResultsA hematoendothelial population was obtained after 84 h of hPSC-EBs formation under serum-free conditions and isolated based on CD144 expression. Intrafemorally injection of hPSC-EB-CD144+ contributed to the generation of CD45+ human cells in immunodeficient mice suggesting the existence of hemogenic ECs within hPSC-EB-CD144+. Endothelial differentiation of hPSC-EB-CD144+ yields a population of > 95% functional ECs in vitro. hPSC-ECs derived through this protocol participated at the formation of new vessels in vivo in a mouse ischemia model. In vitro, hematopoietic differentiation of hPSC-EB-CD144+ generated an intermediate population of > 90% CD43+ hPSC-BCs capable to generate myeloid and erythroid colonies. Finally, the transcriptomic analyses confirmed the hematoendothelial, endothelial and hematopoietic identity of hPSC-EB-CD144+, hPSC-ECs and hPSC-BCs, respectively, and the similarities between hPSC-BC-CD144+CD45+, a subpopulation of hPSC-BCs, and human EL hematopoietic stem cells/hematopoietic progenitors.ConclusionThe present work reports a hPSC differentiation protocol into functional hematopoietic and endothelial cells through a hematoendothelial population. Both lineages were proven to display characteristics of physiological human cells, and therefore, they represent an interesting rapid source of cells for future cell therapy and tissue engineering.

Autofluorescence in freshly isolated adult human liver sinusoidal cells.

Autofluorescent granules of various sizes were observed in primary human liver endothelial cells (LSECs) upon laser irradiation using a wide range of wavelengths. Autofluorescence was detected in LAMP-1 positive vesicles, suggesting lysosomal location. Confocal imaging of freshly prepared cultures and imaging flow cytometry of non-cultured cells revealed fluorescence in all channels used. Treatment with a lipofuscin autofluorescence quencher reduced autofluorescence, most efficiently in the near UV-area. These results, combined with the knowledge of the very active blood clearance function of LSECs support the notion that lysosomally located autofluorescent material reflected accumulation of lipofuscin in the intact liver. These results illustrate the importance of careful selection of exogenous fluorophores, especially when labelling of live cells where the quencher is not compatible.

Autoimmune biomarkers in porto-sinusoidal vascular disease: Potential role in its diagnosis and pathophysiology.

Porto-sinusoidal vascular disease (PSVD) is a rare disease that requires excluding cirrhosis and other causes of portal hypertension for its diagnosis because it lacks a specific diagnostical test. Although it has been occasionally associated with autoimmune diseases, the pathophysiology of PSVD remains unknown. The aim of this study was to evaluate the potential role of autoimmunity in the pathophysiology and diagnosis of PSVD. Thirty-seven consecutive patients with PSVD and 39 with cirrhosis matched by gender, signs of portal hypertension and liver function were included (training set). By using Indirect Immunofluorescence, ELISA and slot-blot methods data 22 autoantibodies were identified in patients with PSVD and cirrhosis. Presence of anti-endothelial cells antibodies (AECA) was assayed by a cell-based ELISA. Thirty-one PSVD, 40 cirrhosis patients, 15 patients with splenomegaly associated with haematological disease and 14 healthy donors were included in a validation set. The proportion of patients with at least one positive antibody was statistically significantly higher in patients with PSVD compared with cirrhosis (92% vs 56%; P<.01). Specifically, AECA were significantly more frequent in PSVD than in cirrhosis (38% vs 15%; P=.013). Results were confirmed in the validation set. In the overall population, presence of AECA had a 63% positive predictive value for diagnosing PSVD and a 71% negative predictive value, with a specificity of 94% when the 1/16 level is used as cut-off. AECA positive serum samples react with a 68-72kDa protein of human liver endothelial sinusoidal cells.

AMIGO2 as a novel indicator of liver metastasis in patients with colorectal cancer.

Our previous study showed that adhesion molecule with immunoglobulin like domain 2 (AMIGO2) is a pivotal driver gene of liver metastasis via regulating tumor cell adhesion to liver endothelial cells in mouse models. The aim of the present study was to clarify the role of AMIGO2 in liver metastasis in patients the colorectal cancer (CRC). Two human CRC cell lines, Caco-2 (AMIGO2-low) and HCT116 (AMIGO2-high), were used in this study. AMIGO2-overexpressing Caco-2 and AMIGO2-knockdown HCT116 cells were generated by transfection with an AMIGO2 expression vector or AMIGO2 small interfering RNA, respectively. Cell proliferation, invasion and adhesion to human liver endothelial cells were examined in in vitro studies. Immunohistochemical analysis was also performed to evaluate the association between AMIGO2 expression and liver metastasis in patients with CRC. In vitro studies revealed that cell proliferation, invasion and adhesion to liver endothelial cells were accelerated by upregulation of AMIGO2 expression, but suppressed by downregulation of AMIGO2 expression in human CRC cells. Immunohistochemical analysis using clinical CRC specimens revealed that AMIGO2 expression was associated with the frequency of liver metastasis (P<0.01), but not that of pulmonary metastasis (P=0.611) and peritoneal dissemination (P=0.909). In addition, AMIGO2 expression levels in tumor cells were significantly higher in liver metastatic foci than primary lesions (P=0.012). In conclusion, the present results indicated that AMIGO2 expression may contribute to the formation of liver metastasis in CRC.

YAP Orchestrates Heterotypic Endothelial Cell Communication via HGF/c-MET Signaling in Liver Tumorigenesis.

The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.

SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells.

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial Cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response.

Investigating the antifibrotic effect of the antiparasitic drug Praziquantel in in vitro and in vivo preclinical models

Tissue fibrosis underlies the majority of human mortality to date with close to half of all reported deaths having a fibrotic etiology. The progression of fibrosis is very complex and reputed irreversible once established. Although some preventive options are being reported, therapeutic options are still scarce and in very high demand, given the rise of diseases linked to fibroproliferative disorders. Our work explored four platforms, complementarily, in order to screen preventive and therapeutic potentials of the antiparasitic drug Praziquantel as a possible antifibrotic. We applied the mouse CCl4-driven liver fibrosis model, the mouse chronic schistosomiasis liver fibrosis model, as well as novel 2D and 3D human cell-based co-culture of human hepatocytes, KCs (Kupffer cells), LECs (Liver Endothelial Cells), HSCs (Hepatic Stellate Cells) and/or myofibroblasts to mimic in vivo fibrotic responses and dynamics. Praziquantel showed some effect on fibrosis marker when preventively administered before severe establishment of fibrosis. However, it failed to potently reverse already established fibrosis. Together, we provided a novel sophisticated multi-assay screening platform to test preventive and therapeutic antifibrotic candidates. We further demonstrated a direct preventive potential of Praziquantel against the onset of fibrosis and the confirmation of its lack of therapeutic potential in reversing already established fibrosis.

HepaChip-MP - a twenty-four chamber microplate for a continuously perfused liver coculture model.

HepaChip microplate (HepaChip-MP) is a microfluidic platform comprised of 24 independent culture chambers with continuous, unidirectional perfusion. In the HepaChip-MP, an automated dielectrophoresis process selectively assembles viable cells into elongated micro tissues. Freshly isolated primary human hepatocytes (PHH) and primary human liver endothelial cells (HuLEC) were successfully assembled as cocultures aiming to mimic the liver sinusoid. Minimal quantities of primary human cells are required to establish micro tissues in the HepaChip-MP. Metabolic function including induction of CYP enzymes in PHH was successfully measured demonstrating a high degree of metabolic activity of cells in HepaChip-MP cultures and sufficient sensitivity of LC-MS analysis even for the relatively small number of cells per chamber. Further, parallelization realized in HepaChip-MP enabled the acquisition of dose-response toxicity data of diclofenac with a single device. Several unique technical features should enable a widespread application of this in vitro model. We have demonstrated fully automated preparation of cell cultures in HepaChip-MP using a pipetting robot. The tubeless unidirectional perfusion system based on gravity-driven flow can be operated within a standard incubator system. Overall, the system readily integrates in workflows common in cell culture labs. Further research will be directed towards optimization of media composition to further extend culture lifetime and study oxygen gradients and their effect on zonation within the sinusoid-like microorgans. In summary, we have established a novel parallelized and scalable microfluidic in vitro liver model showing hepatocyte function and anticipate future in-depth studies of liver biology and applications in pre-clinical drug development.

A human liver cell atlas reveals heterogeneity and epithelial progenitors.

The human liver is an essential multifunctional organ, and liver diseases are rising with limited treatment options. However, the cellular composition of the liver remains poorly understood. Here, we performed single-cell RNA-sequencing of ~10,000 cells from normal liver tissue of 9 human donors to construct a human liver cell atlas. Our analysis revealed previously unknown sub-types among endothelial cells, Kupffer cells, and hepatocytes with transcriptome-wide zonation of some of these populations. We reveal heterogeneity of the EPCAM+ population, which comprises hepatocyte-biased and cholangiocyte populations as well as a TROP2int progenitor population with strong potential to form bipotent liver organoids. As proof-of-principle, we utilized our atlas to unravel phenotypic changes in hepatocellular carcinoma cells and in human hepatocytes and liver endothelial cells engrafted into a mouse liver. Our human liver cell atlas provides a powerful resource enabling the discovery of previously unknown cell types in the normal and diseased liver.

&lt;p&gt;Gold nanoparticles affect the antioxidant status in selected normal human cells&lt;/p&gt;

Purpose: This study evaluates the cytotoxicity of AuNPs coated with polyallylamine (AuNPs-PAA) and conjugated or not to the epidermal growth factor receptor (EGFR)-targeting antibody Cetuximab (AuNPs-PAA-Ctxb) in normal human kidney (HK-2), liver (THLE-2) and microvascular endothelial (TIME) cells, and compares it with two cancer cell lines that are EGFR-overexpressing (A431) or EGFR-negative (MDA-MB-453).Results: Conjugation of Cetuximab to AuNPs-PAA increased the AuNPs-PAA-Ctxb interactions with cells, but reduced their cytotoxicity. TIME cells exhibited the strongest reduction in viability after exposure to AuNPs-PAA(±Ctxb), followed by THLE-2, MDA-MB-453, HK-2 and A431 cells. This cell type-dependent sensitivity was strongly correlated to the inhibition of thioredoxin reductase (TrxR) and glutathione reductase (GR), and to the depolarization of the mitochondrial membrane potential. Both are suggested to initiate apoptosis, which was indeed detected in a concentration- and time-dependent manner. The role of oxidative stress in AuNPs-PAA(±Ctxb)-induced cytotoxicity was demonstrated by co-incubation of the cells with N-acetyl L-cysteine (NAC), which significantly decreased apoptosis and mitochondrial membrane depolarization.Conclusion: This study helps to identify the cells and tissues that could be sensitive to AuNPs and deepens the understanding of the risks associated with the use of AuNPs in vivo.

The Reactive Oxygen Species-Mitophagy Signaling Pathway Regulates Liver Endothelial Cell Survival During Ischemia/Reperfusion Injury.

Ischemia/reperfusion injury (IRI) is the main cause of complications following liver transplantation. Reactive oxygen species (ROS) were thought to be the main regulators of IRI. However, recent studies demonstrate that ROS activate the cytoprotective mechanism of autophagy promoting cell survival. Liver IRI initially damages the liver endothelial cells (LEC), but whether ROS-autophagy promotes cell survival in LEC during IRI is not known. Primary human LEC were isolated from human liver tissue and exposed to an in vitro model of IRI to assess the role of autophagy in LEC. The role of autophagy during liver IRI in vivo was assessed using a murine model of partial liver IRI. During IRI, ROS specifically activate autophagy-related protein (ATG) 7 promoting autophagic flux and the formation of LC3B-positive puncta around mitochondria in primary human LEC. Inhibition of ROS reduces autophagic flux in LEC during IRI inducing necrosis. In addition, small interfering RNA knockdown of ATG7 sensitized LEC to necrosis during IRI. In vivo murine livers in uninjured liver lobes demonstrate autophagy within LEC that is reduced following IRI with concomitant reduction in autophagic flux and increased cell death. In conclusion, these findings demonstrate that during liver IRI ROS-dependent autophagy promotes the survival of LEC, and therapeutic targeting of this signaling pathway may reduce liver IRI following transplantation.

Transmitted/founder hepatitis C viruses induce cell-type- and genotype-specific differences in innate signaling within the liver.

ABSTRACTHepatitis C virus (HCV) infection leads to persistence in the majority of cases despite triggering complex innate immune responses within the liver. Although hepatocytes are the preferred site for HCV replication, nonparenchymal cells (NPCs) can also contribute to antiviral immunity. Recent innovations involving single-genome amplification (SGA), direct amplicon sequencing, and phylogenetic inference have identified full-length transmitted/founder (T/F) viruses. Here, we tested the effect of HCV T/F viral RNA (vRNA) on innate immune signaling within hepatocytes and NPCs, including the HepG2 and Huh 7.5.1 cell lines, a human liver endothelial cell line (TMNK-1), a plasmacytoid dendritic cell line (GEN2.2), and a monocytic cell line (THP-1). Transfection with hepatitis C T/F vRNA induced robust transcriptional upregulation of type I and III interferons (IFNs) within HepG2 and TMNK-1 cells. Both the THP-1 and GEN2.2 lines demonstrated higher type I and III IFN transcription with genotype 3a compared to genotype 1a or 1b. Supernatants from HCV T/F vRNA-transfected TMNK-1 cells demonstrated superior viral control. Primary human hepatocytes (PHH) transfected with genotype 3a induced canonical pathways that included chemokine and IFN genes, as well as overrepresentation of RIG-I (DDX58), STAT1, and a Toll-like receptor 3 (TLR3) network. Full-length molecular clones of HCV induce broad IFN responses within hepatocytes and NPCs, highlighting that signals imparted by the various cell types within the liver may lead to divergent outcomes of infection. In particular, the finding that HCV genotypes differentially induce antiviral responses in NPCs and PHH might account for relevant clinical-epidemiological observations (higher clearance but greater necroinflammation in persistence with genotype 3).