Immune modulation of liver sinusoidal endothelial cells by melittin nanoparticles suppresses liver metastasis.
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ABSTRACT:
SUBMITTER: Yu X
PROVIDER: S-EPMC6361944 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Intracellular gap (iGap) formation in liver sinusoidal endothelial cells (LSECs) is caused by the destruction of fenestrae and appears under pathological conditions; nevertheless, their role in metastasis of cancer cells to the liver remained unexplored. We elucidated that hepatotoxin-damaged and fibrotic livers gave rise to LSECs-iGap formation, which was positively correlated with increased numbers of metastatic liver foci after intrasplenic injection of Hepa1-6 cells. Hepa1-6 cells induced interleukin-23-dependent tumor necrosis factor-α (TNF-α) secretion by LSECs and triggered LSECs-iGap formation, toward which their processes protruded to transmigrate into the liver parenchyma. TNF-α triggered depolymerization of F-actin and induced matrix metalloproteinase 9 (MMP9), intracellular adhesion molecule 1, and CXCL expression in LSECs. Blocking MMP9 activity by doxycycline or an MMP2/9 inhibitor eliminated LSECs-iGap formation and attenuated liver metastasis of Hepa1-6 cells. Overall, this study revealed that cancer cells induced LSEC-iGap formation via proinflammatory paracrine mechanisms and proposed MMP9 as a favorable target for blocking cancer cell metastasis to the liver.
| S-EPMC9519040 | biostudies-literature
Project description:In this study, we developed functionalized polymeric micelles (FPMs) loaded with simvastatin (FPM-Sim) as a drug delivery system to target liver sinusoidal endothelial cells (LSECs) for preserving liver function in chronic liver disease (CLD). Polymeric micelles (PMs) were functionalized by coupling peptide ligands of LSEC membrane receptors CD32b, CD36 and ITGB3. Functionalization was confirmed via spectroscopy and electron microscopy. In vitro and in vivo FPM-Sim internalization was assessed by means of flow cytometry in LSECs, hepatocytes, Kupffer and hepatic stellate cells from healthy rats. Maximum tolerated dose assays were performed in healthy mice and efficacy studies of FPM-Sim were carried out in bile duct ligation (BDL) and thioacetamide (TAA) induction rat models of cirrhosis. Functionalization with the three peptide ligands resulted in stable formulations with a greater degree of in vivo internalization in LSECs than non-functionalized PMs. Administration of FPM-Sim in BDL rats reduced toxicity relative to free simvastatin, albeit with a moderate portal-pressure-lowering effect. In a less severe model of TAA-induced cirrhosis, treatment with FPM-CD32b-Sim nanoparticles for two weeks significantly decreased portal pressure, which was associated with a reduction in liver fibrosis, lower collagen expression as well as the stimulation of nitric oxide synthesis. In conclusion, CD32b-FPM stands out as a good nanotransporter for drug delivery, targeting LSECs, key inducers of liver injury.
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Project description:Tissue-specific endothelial cells are more than simply a barrier lining capillaries and are proved to be capable of remarkable plasticity to become active collagen matrix-producing myofibroblasts (MFs) in solid organs with fibrosis. Liver sinusoidal endothelial cells (LSECs) also participate in the development of hepatic fibrosis, but the exact roles and underlying mechanism have been poorly understood in addition to capillarization. In this study, we demonstrate, by using single-cell RNA sequencing, lineage tracing, and colocalization analysis, that fibrotic LSECs undergo partial endothelial mesenchymal transition (EndMT) with a subset of LSECs acquiring an MF-like phenotype. These phenotypic changes make LSECs substantial producers of extracellular matrix (ECM) preferentially deposited in liver sinusoids but not septal/portal scars as demonstrated by immunofluorescence in animal models and patients with fibrosis/cirrhosis, likely due to their limited migration. Bioinformatic analysis verifies that LSECs undergo successive phenotypic transitions from capillarization to mesenchymal-like cells in liver fibrosis. Furthermore, blockade of LSEC capillarization by using YC-1, a selective eNOS-sGC activator, effectively attenuates liver damage and fibrogenesis as well as mesenchymal features of LSECs, suggesting that capillarization of LSECs might be upstream to their mesenchymal transition during fibrosis. In conclusion, we report that capillarized LSECs undergo a partial EndMT characterized by increased ECM production without activating cell mobility, leading to perisinusoidal ECM deposition that aggravate liver function and fibrogenesis. Targeting this transitional process may be of great value for antifibrotic treatment of liver fibrosis.
| S-EPMC8285099 | biostudies-literature
Project description:Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been analysed. Herein, analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA-20a. Importantly, downregulation of miR-20a occurs in parallel with upregulation of its known protein targets. To restore normal miR-20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate-sorbitan ester nanoparticles conjugated with miR-20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir-20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor-infiltrating capacity and ability of the tumor decreased by approximately 80% in a murine liver metastasis model. Animals: Balb/c mice (6- to 8-week-old males) were obtained from Charles River Laboratories Spain SA (Barcelona, Spain). All the procedures were approved by the Ethical Committee for Animal Experimentation (CEEA) of the University of the Basque Country (EHU/UPV) in accordance with institutional, national and international guidelines regarding the protection and care of animal use for scientific purposes. Mice were kept in the animal facility of EHU/UPV and had access to standard chow and water ad libitum. Colorectal cancer cells: Murine colorectal cancer C26 cells (ATCC, Manassas, VA) syngeneic with Balb/c mice were used. The cells were grown under standard conditions in RPMI medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 microg/ml) and amphotericin B (0.25 microg/ml), all purchased from Life technologies, Carlsbad, CA. Control and tumor-activated LSECs culture: Control and tumour activated primary cultures of LSECs were isolated form livers with colorectal cancer metastasis or from healthy ones. To do so, Balb/c mice were anesthetized with isofluorano and small cut done in their left broadside. Then, 50 microl for each mice of C26 colon carcinoma cells were inoculated in the spleen at 2 x106 cells/ml concentration and the peritoneum and skin were sutured. The control mice were inoculated with PBS 1x buffer. Fourteen days later, all mice were sacrificed and the tumour activated LSECs and control LSECs were isolated by differential centrifugation. To do so, mice were perfused with collagenase P from Clostridium histolyticum (Sigma-Aldrich, St.Louis, MO, USA) through cava vein. The obtained cell suspension was twice centrifuged resulting in a parenchymal (PC)-enriched pellet and a non PC-enriched supernatant. Then, the non PC-enriched supernatant was layered on Percoll gradients (25% on top of 50%) to obtain LSEC. After a centrifugation the interphase between the two density cushions was recollected with purified non-PC enriched LSECs and Kupffer cells (KC). Then, the KCs were separated by adherence and isolated LSECs were washed and cultured with RPMI 1640 medium (Sigma-Aldrich, St.Louis, MO, USA) supplemented with 10% FBS and used in different experiments 24 hours after the isolation. (Life technologies, Carlsbad, CA). miRNA microarray analysis : The purified total RNA was analysed using Agilent mouse miRNA microarrays. Release 18.0, 8x60K (v18) microarray slides (Agilent Technologies, USA), with 1,200 mouse miRNAs represented, were used. Briefly, 100 ng of total RNA was labelled and hybridized following the standard Agilent Protocol for the miRNA Microarray System with a miRNA Complete Labelling and Hybridization Kit, including Agilent miRNA Spike-ins, and the results were scanned using an Agilent Microarray Scanner G2565CA (Agilent Technologies, USA). The scanned TIFF image files were processed using Agilent Feature Extraction Software vs 10.7.3.1 to extract raw data and obtain QC reports. The microarray raw data were normalized using the quantile method. To find the statistically significant differentially expressed miRNAs (DEMs) between two groups of samples, we calculated the mean of each probe across all the samples of the group. Next, we filtered out all the probes whose absolute value of the difference of mean values between the two groups was less than a selection threshold thetaDEM = 4 (that corresponds to a fold change of 2 in log2 scale), and we applied a Student's t-test with a significance threshold alphaDEM = 0.05. The PCA and the hierarchical clustering of genes and samples were performed with one minus the correlation metric and the unweighted average distance (UPGMA) linkage method. All the analysis algorithms and graphics were implemented functions in Matlab.
2021-01-26 | GSE109715 | GEO
Project description:Liver sinusoidal endothelial cells (LSECs) line the low shear, sinusoidal capillary channels of the liver and are the most abundant non-parenchymal hepatic cell population. LSECs do not simply form a barrier within the hepatic sinusoids but have vital physiological and immunological functions, including filtration, endocytosis, antigen presentation and leukocyte recruitment. Reflecting these multifunctional properties, LSECs display unique structural and phenotypic features that differentiate them from the capillary endothelium present within other organs. It is now clear that LSECs have a critical role in maintaining immune homeostasis within the liver and in mediating the immune response during acute and chronic liver injury. In this Review, we outline how LSECs influence the immune microenvironment within the liver and discuss their contribution to immune-mediated liver diseases and the complications of fibrosis and carcinogenesis.
| S-EPMC7096836 | biostudies-literature
Project description:Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that liver sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC-derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.
| S-EPMC7462976 | biostudies-literature
Project description:Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and separate passenger leukocytes in the sinusoidal lumen from hepatocytes. LSECs further act as a platform for adhesion of various liver-resident immune cell populations such as Kupffer cells, innate lymphoid cells or liver dendritic cells. In addition to having an extraordinary scavenger function, LSECs possess potent immune functions, serving as sentinel cells to detect microbial infection through pattern recognition receptor activation and as antigen (cross)-presenting cells. LSECs cross-prime naive CD8 T cells, causing their rapid differentiation into memory T cells that relocate to secondary lymphoid tissues and provide protection when they re-encounter the antigen during microbial infection. Cross-presentation of viral antigens by LSECs derived from infected hepatocytes triggers local activation of effector CD8 T cells and thereby assures hepatic immune surveillance. The immune function of LSECs complements conventional immune-activating mechanisms to accommodate optimal immune surveillance against infectious microorganisms while preserving the integrity of the liver as a metabolic organ.
| S-EPMC4856811 | biostudies-other
Project description:Liver sinusoidal endothelial cells have a gatekeeper function in liver homeostasis by permitting substrates from the bloodstream into the space of Disse and regulating hepatic stellate cell activation status. Maintenance of LSEC's highly specialized phenotype is crucial for liver homeostasis. During liver fibrosis and cirrhosis, LSEC phenotype and functions are lost by processes known as capillarization and LSEC dysfunction. LSEC capillarization can be demonstrated by the loss of fenestrae (cytoplasmic pores) and the manifestation of a basement membrane. Currently, no protein or genetic markers can clearly distinguish healthy from damaged LSECs in acute or chronic liver disease. Single cell (sc)RNA sequencing efforts have identified several LSEC populations in mouse models for liver disease and in human cirrhotic livers. Still, there are no clearly defined genesets that can identify LSECs or dysfunctional LSEC populations in transcriptome data. Here, we developed genesets that are enriched in healthy and damaged LSECs which correlated very strongly with healthy and early stage- vs. advanced human liver diseases. A damaged LSEC signature comprised of Fabp4/5 and Vwf/a1 was established which could efficiently identify damaged endothelial cells in single cell RNAseq data sets. In LSECs from an acute CCl4 liver injury mouse model, Fabp4/5 and Vwf/a1 expression is induced within 1-3 days while in cirrhotic human livers these 4 genes are highly enriched in damaged LSECs. In conclusion, our newly developed gene signature of damaged LSECs can be applicable to a wide range of liver disease etiologies, implicating a common transcriptional alteration mechanism in LSEC damage.
| S-EPMC8564042 | biostudies-literature
Project description:BackgroundLiver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs; liver resident macrophages) form the body's most effective scavenger cell system for the removal of harmful blood-borne substances, ranging from modified self-proteins to pathogens and xenobiotics. Controversies in the literature regarding the LSEC phenotype pose a challenge when determining distinct functionalities of KCs and LSECs. This may be due to overlapping functions of the two cells, insufficient purification and/or identification of the cells, rapid dedifferentiation of LSECs in vitro, or species differences. We therefore characterized and quantitatively compared expressed gene products of freshly isolated, highly pure LSECs (fenestrated SE-1/FcγRIIb2+) and KCs (CD11b/c+) from Sprague Dawley, Crl:CD (SD), male rats using high throughput mRNA-sequencing and label-free proteomics.ResultsWe observed a robust correlation between the proteomes and transcriptomes of the two cell types. Integrative analysis of the global molecular profile demonstrated the immunological aspects of LSECs. The constitutive expression of several immune genes and corresponding proteins of LSECs bore some resemblance with the expression in macrophages. LSECs and KCs both expressed high levels of scavenger receptors (SR) and C-type lectins. Equivalent expression of SR-A1 (Msr1), mannose receptor (Mrc1), SR-B1 (Scarb1), and SR-B3 (Scarb2) suggested functional similarity between the two cell types, while functional distinction between the cells was evidenced by LSEC-specific expression of the SRs stabilin-1 (Stab1) and stabilin-2 (Stab2), and the C-type lectins LSECtin (Clec4g) and DC-SIGNR (Clec4m). Many immune regulatory factors were differentially expressed in LSECs and KCs, with one cell predominantly expressing a specific cytokine/chemokine and the other cell the cognate receptor, illustrating the complex cytokine milieu of the sinusoids. Both cells expressed genes and proteins involved in antigen processing and presentation, and lymphocyte co-stimulation.ConclusionsOur findings support complementary and partly overlapping scavenging and immune functions of LSECs and KCs. This highlights the importance of including LSECs in studies of liver immunity, and liver clearance and toxicity of large molecule drugs and nano-formulations.
| S-EPMC7694354 | biostudies-literature
Project description:The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease.
| S-EPMC3119346 | biostudies-literature