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Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

ABSTRACT: The pivotal role of hepatic stellate cells (HSCs) in orchestrating the bidirectional process of progression and regression of liver fibrosis makes them an ideal target for exploring new antifibrotic therapies. Essential phospholipids (EPLs), with their polyenylphosphatidylcholine (PPC) fraction, either alone or combined with other hepatoprotective substances such as silymarin, are recommended in hepatic impairment, but a scientific rationale for their use is still lacking. Herein, we compared the ability of EPLs to restore quiescent-like features in HSCs with that of dilinoleoylphosphatidylcholine (DLPC), PPC fraction's main component. Specifically, we screened at the cellular level the antifibrotic effects of PPC formulations in the presence and absence of silymarin, by using LX-2 cells (pro-fibrogenic HSCs) and by assessing the main biochemical hallmarks of the activated and deactivated states of this cell line. We also proved the formulations' direct effect on the motional order of cell membranes of adherent cells. LX-2 cells, examined for lipid droplets as a quiescence marker, showed that PPCs led to a more prominent deactivation than DLPC. This result was confirmed by a reduction of collagen and ?-SMA expression, and by a profound alteration in the cell membrane fluidity. PPC-silymarin formulations deactivated HSCs with a significant synergistic effect. The remarkable bioactivity of PPCs in deactivating fibrogenic HSCs paves the way for the rational design of new therapeutics aimed at managing hepatic fibrosis.

SUBMITTER: Valentino G

PROVIDER: S-EPMC6969915 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

Valentino Gina G Zivko Cristina C Weber Florian F Brülisauer Lorine L Luciani Paola P

Pharmaceutics 20191212 12

The pivotal role of hepatic stellate cells (HSCs) in orchestrating the bidirectional process of progression and regression of liver fibrosis makes them an ideal target for exploring new antifibrotic therapies. Essential phospholipids (EPLs), with their polyenylphosphatidylcholine (PPC) fraction, either alone or combined with other hepatoprotective substances such as silymarin, are recommended in hepatic impairment, but a scientific rationale for their use is still lacking. Herein, we compared th ...[more]

PMID: 31842373

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Project description:Background & aimsActivation of Kupffer cells and recruitment of monocytes are key events in fibrogenesis. These cells release soluble mediators which induce the activation of hepatic stellate cells (HSCs), the main fibrogenic cell type within the liver. Mer tyrosine kinase (MerTK) signaling regulates multiple processes in macrophages and has been implicated in the pathogenesis of non-alcoholic steatohepatitis-related fibrosis. In this study, we explored if MerTK activation in macrophages influences the profibrogenic phenotype of HSCs.MethodsMacrophages were derived from THP-1 cells or differentiated from peripheral blood monocytes towards MerTK+/CD206+/CD163+/CD209- macrophages. The role of MerTK was assessed by pharmacologic and genetic inhibition. HSC migration was determined in Boyden chambers, viability was measured by the MTT assay, and proliferation was evaluated by the BrdU incorporation assay.ResultsGas-6 induced MerTK phosphorylation and Akt activation in macrophages, and these effects were inhibited by UNC569. During polarization, MerTK+/CD206+/CD163+/CD209- macrophages exhibited activation of STAT3, ERK1/2, p38 and increased expression of VEGF-A. Activation of MerTK in THP-1 macrophages induced a secretome which promoted a significant increase in migration, proliferation, viability and expression of profibrogenic factors in HSCs. Similarly, conditioned medium from MerTK+ macrophages induced a significant increase in cell migration, proliferation, STAT3 and p38 phosphorylation and upregulation of IL-8 expression in HSCs. Moreover, conditioned medium from Gas-6-stimulated Kupffer cells induced a significant increase in HSC proliferation. These effects were specifically related to MerTK expression and activity in macrophages, as indicated by pharmacologic inhibition and knockdown experiments.ConclusionsMerTK activation in macrophages modifies the secretome to promote profibrogenic features in HSCs, implicating this receptor in the pathogenesis of hepatic fibrosis.Lay summaryFibrosis represents the process of scarring occurring in patients with chronic liver diseases. This process depends on production of scar tissue components by a specific cell type, named hepatic stellate cells, and is regulated by interaction with other cells. Herein, we show that activation of MerTK, a receptor present in a population of macrophages, causes the production of factors that act on hepatic stellate cells, increasing their ability to produce scar tissue.

Project description:Background and aimsAntifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis.Approach and resultsCygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-β secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers.ConclusionsHis-CYGB could have antifibrotic clinical applications for human chronic liver diseases.

Project description:BackgroundLiver fibrosis is characterized by the excessive formation and accumulation of matrix proteins as a result of wound healing in the liver. A main event during fibrogenesis is the activation of the liver resident quiescent hepatic stellate cell (qHSC). Recent studies suggest that reversion of the activated HSC (aHSC) phenotype into a quiescent-like phenotype could be a major cellular mechanism underlying fibrosis regression in the liver, thereby offering new therapeutic perspectives for the treatment of liver fibrosis. Whether human HSCs have the ability to undergo a similar reversion in phenotype is currently unknown. The aim of the present study is to identify experimental conditions that can revert the in vitro activated phenotype of primary human HSCs and consequently to map the molecular events associated with this reversion process by gene expression profiling.ResultsWe find that epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2) synergistically downregulate the expression of ACTA2 and LOX in primary human aHSCs. Their combination with oleic acid, palmitic acid, and retinol further potentiates a more quiescent-like phenotype as demonstrated by the abundant presence of retinyl ester-positive intra-cytoplasmic lipid droplets, low expression levels of activation markers, and a reduced basal as well as cytokine-stimulated proliferation and matrix metalloproteinase activity. Gene expression profiling experiments reveal that these in vitro reverted primary human HSCs (rHSCs) display an intermediary phenotype that is distinct from qHSCs and aHSCs. Interestingly, this intermediary phenotype is characterized by the increased expression of several previously identified signature genes of in vivo inactivated mouse HSCs such as CXCL1, CXCL2, and CTSS, suggesting also a potential role for these genes in promoting a quiescent-like phenotype in human HSCs.ConclusionsWe provide evidence for the ability of human primary aHSCs to revert in vitro to a transitional state through synergistic action of EGF, FGF2, dietary fatty acids and retinol, and provide a first phenotypic and genomic characterization of human in vitro rHSCs.

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Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

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Synergy of Phospholipid-Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells.

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