The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report ...the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro.
Primary sclerosing cholangitis (PSC) is a rare, progressive disease, characterized by inflammation and fibrosis of the bile ducts, lacking reliable prognostic biomarkers for disease activity. Machine ...learning applied to broad proteomic profiling of sera allowed for the discovery of markers of disease presence, severity, and cirrhosis and the exploration of the involvement of CCL24, a chemokine with fibro-inflammatory activity. Sera from 30 healthy controls and 45 PSC patients were profiled with proximity extension assay, quantifying the expression of 2870 proteins, and used to train an elastic net model. Proteins that contributed most to the model were tested for correlation to enhanced liver fibrosis (ELF) score and used to perform pathway analysis. Statistical modeling for the presence of cirrhosis was performed with principal component analysis (PCA), and receiver operating characteristics (ROC) curves were used to assess the useability of potential biomarkers. The model successfully predicted the presence of PSC, where the top-ranked proteins were associated with cell adhesion, immune response, and inflammation, and each had an area under receiver operator characteristic (AUROC) curve greater than 0.9 for disease presence and greater than 0.8 for ELF score. Pathway analysis showed enrichment for functions associated with PSC, overlapping with pathways enriched in patients with high levels of CCL24. Patients with cirrhosis showed higher levels of CCL24. This data-driven approach to characterize PSC and its severity highlights potential serum protein biomarkers and the importance of CCL24 in the disease, implying its therapeutic potential in PSC.
Background & Aims Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in children and adolescents in industrialized countries. It is important to accurately determine ...the stage of fibrosis in these patients. The enhanced liver fibrosis (ELF) test has been validated for staging liver fibrosis in adult patients with chronic liver diseases, including NAFLD. We investigated the performance of this test in assessing liver fibrosis in children and adolescents with NAFLD, identified by biopsy. Methods The ELF test was performed on a panel of serum samples collected from 112 consecutive subjects that were likely to have NAFLD (64 male, mean age of 13.8 ± 3.3). A previously described and validated algorithm was used to analyze the data on hyaluronic acid (HA), amino-terminal propeptide of type III collagen (PIIINP), and tissue inhibitor of metalloproteinase 1 (TIMP-1) levels. Results In pediatric patients with NAFLD, the ELF test predicted liver fibrosis stage with a high degree of sensitivity and specificity; results were superior to those reported for adults. The area under receiver operating characteristic curves/best possible ELF test cut-off values for the prediction of “any” (≥stage 1), moderate-perisinusoidal (≥stage 1b), moderate-portal/periportal (≥stage 1c), significant (≥stage 2), or advanced (≥stage 3) fibrosis were 0.92/9.28, 0.92/9.33, 0.90/9.54, 0.98/10.18 and 0.99/10.51, respectively. Conclusions The ELF test can be used to accurately assess the level of liver fibrosis in pediatric patients with NAFLD. This information is important for identifying patients with progressive fibrosis that require further histopathological analysis or therapeutic follow-up.
Background & Aims:
Histologic examination of a liver biopsy specimen is regarded as the reference standard for detecting liver fibrosis. Biopsy can be painful and hazardous, and assessment is ...subjective and prone to sampling error. We developed a panel of sensitive automated immunoassays to detect matrix constituents and mediators of matrix remodeling in serum to evaluate their performance in the detection of liver fibrosis.
Methods:
In an international multicenter cohort study, serum levels of 9 surrogate markers of liver fibrosis were compared with fibrosis stage in liver biopsy specimens obtained from 1021 subjects with chronic liver disease. Discriminant analysis of a test set of samples was used to identify an algorithm combining age, hyaluronic acid, amino-terminal propeptide of type III collagen, and tissue inhibitor of matrix metalloproteinase 1 that was subsequently evaluated using a validation set of biopsy specimens and serum samples.
Results:
The algorithm detected fibrosis (sensitivity, 90%) and accurately detected the absence of fibrosis (negative predictive value for significant fibrosis, 92%; area under the curve of a receiver operating characteristic plot, .804; standard error, .02;
P < .0001; 95% confidence interval, .758–.851). Performance was excellent for alcoholic liver disease and nonalcoholic fatty liver disease. The algorithm performed equally well in comparison with each of the pathologists. In contrast, pathologists’ agreement over histologic scores ranged from very good to moderate (κ = .97–.46).
Conclusions:
Assessment of liver fibrosis with multiple serum markers used in combination is sensitive, specific, and reproducible, suggesting they may be used in conjunction with liver biopsy to assess a range of chronic liver diseases.
Following acute or chronic liver tissue damage, hepatic stellate cells (HSCs) undergo a process of activation toward a phenotype characterized by increased proliferation, motility, contractility, and ...synthesis of extracellular matrix components. Activation of HSCs is regulated by several soluble factors, including growth factors, cytokines, chemokines, and products of oxidative stress, as well as by extensive changes in the composition and organization of the ECM. Different groups of soluble factors may be classified according to their prevalent biological effect: (a) factors promoting HSC proliferation and/or migration (i.e., platelet-derived growth factor, basic fibroblast growth factor, insulin-like growth factor-1); (b) factors promoting fibrillar ECM accumulation, particularly transforming growth factor-beta1; (c) factors with a prevalent contractile effect on HSCs, such as endothelin-1, thrombin, angiotensin-II and vasopressin, although all these agents also may promote HSC proliferation; (d) proinflammatory cytokines and chemokines; and (e) cytokines with a prominent antiinflammatory/antifibrogenic activity, such as interleukin-10 and interferon-gamma. Additional important issues are represented by the relationship between cytokine and integrin signaling, and by the effects of oxidative stress-related molecules on cytokine signaling. In the past decade the major intracellular signaling pathways elicited by these factors in HSCs have been greatly elucidated.
Hepatic tissue engineering using decellularized scaffolds is a potential therapeutic alternative to conventional transplantation. However, scaffolds are usually obtained using decellularization ...protocols that destroy the extracellular matrix (ECM) and hamper clinical translation. We aim to develop a decellularization technique that reliably maintains hepatic microarchitecture and ECM components. Isolated rat livers were decellularized by detergent-enzymatic technique with (EDTA-DET) or without EDTA (DET). Histology, DNA quantification and proteomics confirmed decellularization with further DNA reduction with the addition of EDTA. Quantification, histology, immunostaining, and proteomics demonstrated preservation of extracellular matrix components in both scaffolds with a higher amount of collagen and glycosaminoglycans in the EDTA-DET scaffold. Scanning electron microscopy and X-ray phase contrast imaging showed microarchitecture preservation, with EDTA-DET scaffolds more tightly packed. DET scaffold seeding with a hepatocellular cell line demonstrated complete repopulation in 14 days, with cells proliferating at that time. Decellularization using DET preserves microarchitecture and extracellular matrix components whilst allowing for cell growth for up to 14 days. Addition of EDTA creates a denser, more compact matrix. Transplantation of the scaffolds and scaling up of the methodology are the next steps for successful hepatic tissue engineering.
Cytoglobin (CYGB) belongs to the mammalian globin family and is exclusively expressed in hepatic stellate cells (HSCs) in the liver. In addition to its gas-binding ability, CYGB is relevant to ...hepatic inflammation, fibrosis, and cancer because of its anti-oxidative properties; however, the regulation of CYGB gene expression remains unknown. Here, we sought to identify factors that induce CYGB expression in HSCs and to clarify the molecular mechanism involved. We used the human HSC cell line HHSteC and primary human HSCs isolated from intact human liver tissues. In HHSteC cells, treatment with a culture supplement solution that included fibroblast growth factor 2 (FGF2) increased CYGB expression with concomitant and time-dependent α-smooth muscle actin (αSMA) down-regulation. We found that FGF2 is a key factor in inducing the alteration in both CYGB and αSMA expression in HHSteCs and primary HSCs and that FGF2 triggered the rapid phosphorylation of both c-Jun N-terminal kinase (JNK) and c-JUN. Both the JNK inhibitor PS600125 and transfection of c-JUN–targeting siRNA abrogated FGF2-mediated CYGB induction, and conversely, c-JUN overexpression induced CYGB and reduced αSMA expression. Chromatin immunoprecipitation analyses revealed that upon FGF2 stimulation, phospho-c-JUN bound to its consensus motif (5′-TGA(C/G)TCA), located −218 to −222 bases from the transcription initiation site in the CYGB promoter. Of note, in bile duct–ligated mice, FGF2 administration ameliorated liver fibrosis and significantly reduced HSC activation. In conclusion, FGF2 triggers CYGB gene expression and deactivation of myofibroblastic human HSCs, indicating that FGF2 has therapeutic potential for managing liver fibrosis.
Background and Aims
Antifibrotic 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 Results
Cygb‐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.
Conclusions
His‐CYGB could have antifibrotic clinical applications for human chronic liver diseases.
The prognostic role of spontaneous portosystemic shunts (SPSS) has been poorly investigated.
To evaluate the impact of the presence of SPSS, as well as their characteristics, on the risk of ...decompensation.
This is a retrospective cohort study of 235 advanced chronic liver disease (ACLD) patients with available imaging examination, transient elastography, and upper endoscopy. ACLD was defined as liver stiffness measurement (LSM) >10 kPa. Competitive risk analyses were performed to identify the factors associated with the main outcome.
SPSS were reported in 141 (60%) of the patients. Non-viral etiology was independently associated with SPSS presence Odds-Ratio (OR): 2.743;95%-Interval-of-Confidence (IC):1.129–6.664. During a follow-up of 37 (20–63) months, SPSS were found predictors of any decompensation type Subhazard Ratio (SHR):2.264; 95%-IC:1.259–4.071, independently from a history of decompensation or high-risk-varices presence. The risk of complications was higher in patients with large (SHR: 3.775; 95%-IC: 2.016–7.070) and multiple (SHR:3.832; 95%-IC: 2.004–7.330) shunts, and in those with gastrorenal shunts (SHR:2.636; 95%-IC:1.521–4.569).
The presence, size, and number of SPSS predict not only the risk of hepatic encephalopathy but that of any type of decompensation across all stages of cirrhosis. Future studies should explore the possibility of treating shunts to prevent decompensation.
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