Hepatic stellate cells that coexpress LRAT and CRBP-1 partially contribute to portal fibrogenesis in patients with human viral hepatitis.

BACKGROUND & AIMS: Precisely what type of cells mainly contributes to portal fibrosis, especially in chronic viral hepatitis, such as hepatic stellate cells (HSCs) in the parenchyma or myofibroblasts in the portal area, still remains unclear. It is necessary to clarify the characteristics of cells that contribute to portal fibrosis in order to determine the mechanism of portal fibrogenesis and to develop a therapeutic target for portal fibrosis. This study was undertaken to examine whether LRAT+/CRBP-1+ HSCs contribute to portal fibrosis on viral hepatitis. METHODS: Antibodies to lecithin:retinol acyltransferase (LRAT), cellular retinol-binding protein-1 (CRBP-1) and widely ascertained antibodies to HSCs (alpha-smooth muscle actin, neurotrophin-3) and endothelial cells (CD31) were used for immunohistochemical studies to assess the distribution of cells that contribute to the development of portal fibrosis with the aid of fluorescence microscopy. A quantitative analysis of LRAT+/CRBP-1+ HSCs was performed. RESULTS: The number of LRAT+/CRBP-1+ HSCs was increased in fibrotic liver in comparison with normal liver in the portal area and fibrous septa. The number of double positive cells was less than 20% of all cells/field in maximum. CONCLUSION: This study provides evidence that functional HSCs coexpressing both LRAT and CRBP-1 that continue to maintain the ability to store vitamin A contribute in part to the development of portal fibrogenesis in addition to parenchymal fibrogenesis in patients with viral hepatitis.

Characterization of human stem cell-derived hepatic stellate cells and liver sinusoidal endothelial cells during extended in vitro culture.

Background: There is a significant need for predictive and stable in vitro human liver representations for disease modeling and drug testing. Hepatic stellate cells (HSCs) and liver sinusoidal endothelial cells (LSECs) are important non-parenchymal cell components of the liver and are hence of relevance in a variety of disease models, including hepatic fibrosis. Pluripotent stem cell- (PSC-) derived HSCs (scHSCs) and LSECs (scLSECs) offer an attractive alternative to primary human material; yet, the suitability of scHSCs and scLSECs for extended in vitro modeling has not been characterized. Methods: In this study, we describe the phenotypic and functional development of scHSCs and scLSECs during 14 days of 2D in vitro culture. Cell-specific phenotypes were evaluated by cell morphology, immunofluorescence, and gene- and protein expression. Functionality was assessed in scHSCs by their capacity for intracellular storage of vitamin A and response to pro-fibrotic stimuli induced by TGF-ß. scLSECs were evaluated by nitric oxide- and factor VIII secretion as well as endocytic uptake of bioparticles and acetylated low-density lipoprotein. Notch pathway inhibition and co-culturing scHSCs and scLSECs were separately tested as options for enhancing long-term stability and maturation of the cells. Results and Conclusion: Both scHSCs and scLSECs exhibited a post-differentiation cell type-specific phenotype and functionality but deteriorated during extended culture with PSC line-dependent variability. Therefore, the choice of PSC line and experimental timeframe is crucial when designing in vitro platforms involving scHSCs and scLSECs. Notch inhibition modestly improved long-term monoculture in a cell line-dependent manner, while co-culturing scHSCs and scLSECs provides a strategy to enhance phenotypic and functional stability.

The Extra Virgin Olive Oil Polyphenol Oleocanthal Exerts Antifibrotic Effects in the Liver.

Liver fibrosis, which is the outcome of wound-healing response to chronic liver damage, represents an unmet clinical need. This study evaluated the anti-fibrotic and anti-inflammatory effects of the polyphenol oleocanthal (OC) extracted from extra virgin olive oil (EVOO) by an in vitro/in vivo approach. The hepatic cell lines LX2 and HepG2 were used as in vitro models. The mRNA expression of pro-fibrogenic markers, namely alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1), a panel of metalloproteinases (MMP1, MMP2, MMP3, MMP7, MMP9) and vascular endothelial growth factor A (VEGFA) as well as the pro-oxidant genes NADPH oxidases (NOXs) 1 and 4 were evaluated in TGF-ß activated LX2 cells by qRT-PCR. α-SMA and COL1A1 protein expression was assessed by immunofluorescence coupled to confocal microscopy. VEGFA release from LX2 was measured by ELISA. We also evaluated the amount of reactive oxygen species (ROS) produced by H2O2 activated- HepG2 cells. In vivo, OC was administered daily by oral gavage to Balb/C mice with CCl4-induced liver fibrosis. In this model, we measured the mRNA hepatic expression of the three pro-inflammatory interleukins (IL) IL6, IL17, IL23, chemokines such as C-C Motif Chemokine Ligand 2 (CCL2) and C-X-C Motif Chemokine Ligand 12 (CXCL12), and selected miRNAs (miR-181-5p, miR-221-3p, miR-29b-3p and miR-101b-3p) by qRT-PCR. We demonstrated that OC significantly downregulated the gene/protein expression of α-SMA, COL1A1, MMP2, MMP3, MMP7 and VEGF as well as the oxidative enzymes NOX1 and 4 in TGFß1-activated LX2 cells, and reduced the production of ROS by HepG2. In vivo OC, beside causing a significant reduction of fibrosis at histological assessment, counteracted the CCl4-induced upregulation of pro-fibrotic and inflammatory genes. Moreover, OC upregulated the anti-fibrotic miRNAs (miR-29b-3p and miR-101b-3p) reduced in fibrotic mice, while downregulated the pro-fibrotic miRNAs (miR-221-3p and miR-181-5p), which were dramatically upregulated in fibrotic mice. In conclusion, OC exerts a promising antifibrotic effect via a combined reduction of oxidative stress and inflammation involving putative miRNAs, which in turn reduces hepatic stellate cells activation and liver fibrosis.