A rule-based multiscale model of hepatic stellate cell plasticity: Critical role of the inactivation loop in fibrosis progression.

Hepatic stellate cells (HSC) are the source of extracellular matrix (ECM) whose overproduction leads to fibrosis, a condition that impairs liver functions in chronic liver diseases. Understanding the dynamics of HSCs will provide insights needed to develop new therapeutic approaches. Few models of hepatic fibrosis have been proposed, and none of them include the heterogeneity of HSC phenotypes recently highlighted by single-cell RNA sequencing analyses. Here, we developed rule-based models to study HSC dynamics during fibrosis progression and reversion. We used the Kappa graph rewriting language, for which we used tokens and counters to overcome temporal explosion. HSCs are modeled as agents that present seven physiological cellular states and that interact with (TGFß1) molecules which regulate HSC activation and the secretion of type I collagen, the main component of the ECM. Simulation studies revealed the critical role of the HSC inactivation process during fibrosis progression and reversion. While inactivation allows elimination of activated HSCs during reversion steps, reactivation loops of inactivated HSCs (iHSCs) are required to sustain fibrosis. Furthermore, we demonstrated the model's sensitivity to (TGFß1) parameters, suggesting its adaptability to a variety of pathophysiological conditions for which levels of (TGFß1) production associated with the inflammatory response differ. Using new experimental data from a mouse model of CCl4-induced liver fibrosis, we validated the predicted ECM dynamics. Our model also predicts the accumulation of iHSCs during chronic liver disease. By analyzing RNA sequencing data from patients with non-alcoholic steatohepatitis (NASH) associated with liver fibrosis, we confirmed this accumulation, identifying iHSCs as novel markers of fibrosis progression. Overall, our study provides the first model of HSC dynamics in chronic liver disease that can be used to explore the regulatory role of iHSCs in liver homeostasis. Moreover, our model can also be generalized to fibroblasts during repair and fibrosis in other tissues.

Characterization of hepatitis B viral forms from patient plasma using velocity gradient: Evidence for an excess of capsids in fractions enriched in Dane particles.

Hepatitis B virus (HBV) morphogenesis is characterized by a large over-production of subviral particles and recently described new forms in parallel of complete viral particles (VP). This study was designed to depict circulating viral forms in HBV infected patient plasmas, using velocity gradients and most sensitive viral markers. Plasmas from chronic hepatitis B (CHB) patients, HBeAg positive or negative, genotype D or E, were fractionated on velocity and equilibrium gradients with or without detergent treatment. Antigenic and molecular markers were measured in plasma and in each collected fraction. Fast Nycodenz velocity gradients revealed good reproducibility and provided additional information to standard equilibrium sucrose gradients. HBV-RNAs circulated as enveloped particles in all plasmas, except one, and at lesser concentrations than VP. Calculations based on standardized measurements and relative virion and subviral particle molecular stoichiometry allowed to refine the experimental approach. For the HBeAg-positive plasma, VP were accompanied by an overproduction of enveloped capsids, either containing HBs, likely corresponding to empty virions, or for the main part, devoid of this viral envelope protein. Similarly, in the HBeAg-negative sample, HBs enveloped capsids, likely corresponding to empty virions, were detected and the presence of enveloped capsids devoid of HBs protein was suspected but not clearly evidenced due to the presence of contaminating high-density subviral particles. While HBeAg largely influences HBcrAg measurement and accounts for two-thirds of HBcrAg reactivity in HBeAg-positive patients, it remains a 10 times more sensitive marker than HBsAg to characterize VP containing fractions. Using Nycodenz velocity gradients and standardized biomarkers, our study proposes a detailed characterization of circulating viral forms in chronically HBV infected patients. We provide evidence for an excess of capsids in fractions enriched in Dane particles, likely due to the presence of empty virions but also by capsids enveloped by an HBs free lipid layer. Identification of this new circulating viral particle sets the basis for studies around the potential role of these entities in hepatitis B pathogeny and their physiological regulation.

The contribution of more sensitive hepatitis B surface antigen assays to detecting and monitoring hepatitis B infection.

BACKGROUND: Hepatitis B surface antigen (HBsAg) remains the main viral marker for screening and monitoring hepatitis B virus (HBV) infection. The quantification limit of most current HBsAg assays is around 0.05 IU/mL. The Lumipulse-G-HBsAg-Quant assay (Fujirebio) claims to obtain a tenfold improvement in sensitivity. This study aimed to assess the performance of this assay in detecting low HBsAg levels in clinical samples. METHODS: Three panels of stored frozen samples were selected on the basis of HBV-DNA and HBsAg values obtained previously with routine techniques. Panels 1 (n=13) and 2 (n=52) consisted of DNA-positive/HBsAg-negative samples from individuals in the window period and with occult HBV infection respectively. Panel 3 comprised 23 samples with low or discrepant HBsAg screening results. All these samples were tested retrospectively with the DiaSorin and Fujirebio HBsAg assays. RESULTS: Sixteen out of 65 samples (25 %), initially screened HBsAg negative, were reactive only with the Fujirebio assay (median value= 0.015 IU/mL; IQR= 0.012): three (23 %) samples from panel 1 and 13 (25 %) from panel 2. Thirteen of these 16 (81 %) had HBsAg values below 0.03 IU/mL with the DiaSorin assay. In panel 3, 22 (96 %) samples were quantified successfully with the Fujirebio assay (median: 0.32 IU/mL; IQR: 1.20) and 19 (83 %) with the DiaSorin assay (median: 0.31 IU/mL; IQR: 0.65). Concentrations obtained with the two assays showed good correlations (r=0.893, Spearman). CONCLUSIONS: HBsAg assays with enhanced analytical sensitivity could improve HBV serological profile interpretation with possible consequences on clinical management of infected patients, and on blood transfusion safety.

Increasing 3D Matrix Rigidity Strengthens Proliferation and Spheroid Development of Human Liver Cells in a Constant Growth Factor Environment.

Mechanical forces influence the growth and shape of virtually all tissues and organs. Recent studies show that increased cell contractibility, growth and differentiation might be normalized by modulating cell tensions. Particularly, the role of these tensions applied by the extracellular matrix during liver fibrosis could influence the hepatocarcinogenesis process. The objective of this study is to determine if 3D stiffness could influence growth and phenotype of normal and transformed hepatocytes and to integrate extracellular matrix (ECM) stiffness to tensional homeostasis. We have developed an appropriate 3D culture model: hepatic cells within three-dimensional collagen matrices with varying rigidity. Our results demonstrate that the rigidity influenced the cell phenotype and induced spheroid clusters development whereas in soft matrices, Huh7 transformed cells were less proliferative, well-spread and flattened. We confirmed that ERK1 played a predominant role over ERK2 in cisplatin-induced death, whereas ERK2 mainly controlled proliferation. As compared to 2D culture, 3D cultures are associated with epithelial markers expression. Interestingly, proliferation of normal hepatocytes was also induced in rigid gels. Furthermore, biotransformation activities are increased in 3D gels, where CYP1A2 enzyme can be highly induced/activated in primary culture of human hepatocytes embedded in the matrix. In conclusion, we demonstrated that increasing 3D rigidity could promote proliferation and spheroid developments of liver cells demonstrating that 3D collagen gels are an attractive tool for studying rigidity-dependent homeostasis of the liver cells embedded in the matrix and should be privileged for both chronic toxicological and pharmacological drug screening.

Linear least square (LLS) method for pixel-resolution analysis of polarization dependent SHG images of collagen fibrils.

A linear least square (LLS) method is proposed to process polarization dependent SHG intensity analysis at pixel-resolution level in order to provide an analytic solution of nonlinear susceptibility χ(2) coefficients and of fibril orientation. This model is applicable to fibrils with identical orientation in the excitation volume. It has been validated on type I collagen fibrils from cell-free gel, tendon and extracellular matrix of F1 biliary epithelial cells. LLS is fast (a few hundred milliseconds for a 512 × 512 pixel image) and very easy to perform for non-expert in numerical signal processing. Theoretical simulation highlights the importance of signal to noise ratio for accurate determination of nonlinear susceptibility χ(2) coefficients. The results also suggest that, in addition to the peptide group, a second molecular nonlinear optical hyperpolarizability ß contributes to the SHG signal. Finally from fibril orientation analysis, results show that F1 cells remodel extracellular matrix collagen fibrils by changing fibril orientation, which might have important physiological function in cell migration and communication.

Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42-Tuba pathway.

Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. We previously found that collagen I fibrils induced the formation of peculiar linear invadosomes in an unexpected integrin-independent manner. Here, we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer, colocalizes with linear invadosomes in tumor cells and is required for their formation and matrix degradation ability. Unexpectedly, DDR1 kinase activity is not required for invadosome formation or activity, nor is Src tyrosine kinase. We show that the RhoGTPase Cdc42 is activated on collagen in a DDR1-dependent manner. Cdc42 and its specific guanine nucleotide-exchange factor (GEF), Tuba, localize to linear invadosomes, and both are required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasion in a collagen gel. Altogether, our data uncover an important role for DDR1, acting through Tuba and Cdc42, in proteolysis-based cell invasion in a collagen-rich environment.